Peptides that specifically bind hgf receptor (cmet) and uses thereof

ABSTRACT

A polypeptide or multimeric polypeptide construct having the ability to bind to cMet or a complex comprising cMet and HGF, and methods for use are disclosed.

This application claims priority under 35 USC §119(e) to U.S.Provisional Patent Application Ser. No. 60/451,588, filed on Mar. 3,2003, the entire contents of which is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Hepatocyte growth factor (also known as scatter factor) is amulti-functional growth factor involved in various physiologicalprocesses such as embryogenesis, wound healing and angiogenesis. It hasbecome apparent that HGF, through interactions with its high affinityreceptor (cMet), is involved in tumor growth, invasion and metastasis.In fact, dysregulated cMet expression (for example, the overexpressionof cMet in neoplastic epithelium of colorectal adenomas and in othercarcinomas as compared to normal mucosa) and/or activity, as well ashyperactivity of the cMet receptor through an autocrine stimulatory loopwith HGF, has been demonstrated in a variety of tumor tissues andinduces oncogenic transformation of specific cell lines.

In general, HGF is produced by the stromal cells, which form part ofmany epithelial tumors; however, it is believed that the production ofHGF by tumor cells themselves comprises the main pathway leading to thehyperproliferation of specific tumors. HGF/cMet autocrine stimulatoryloops have been detected in, gliomas, osteosarcomas, and mammary,prostate, breast, lung and other carcinomas.

Interrupting the HGF interaction with the cMet receptor slows tumorprogression in animal models. In addition to stimulating proliferationof certain cancer cells through activation of cMet, HGF also protectsagainst DNA-damaging agent-induced cytotoxicity in a variety of celllines susceptible to hyperproliferative phenotypes (e.g., breastcancer). Therefore, preventing HGF from binding to cMet could predisposecertain cancer cells to the cytotoxicity of certain drugs.

In addition to hyperproliferative disorders, cMet also has been linkedto angiogenesis. For example, stimulation of cMet leads to theproduction of vascular endothelial growth factor (VEGF), which, in turn,stimulates angiogenesis. Additionally, stimulation of cMet also has beenimplicated in promoting wound healing.

In addition to identifying the cMet receptor as a therapeutic target forhyperproliferative disorders, angiogenesis and wound healing, the largediscrepancy between expression levels of neoplastic and correspondingnormal tissues indicates that cMet is an attractive target for imagingapplications directed to hyperproliferative disorders.

SUMMARY OF THE INVENTION

The present invention relates to peptides, peptide complexes andcompositions having the ability to bind to cMet and antagonizehepatocyte growth factor (HGF) activity by preventing HGF from bindingto cMet. In addition, this invention relates to such peptides, peptidecomplexes and compositions having the ability to bind to cMet for thepurpose of detecting and targeting this receptor, inhibiting cMetactivity independent of HGF antagonistic properties, and for the purposeof diagnostic imaging. The involvement of the HGF/cMet axis in a varietyof cellular functions including cellular proliferation, wound healingand angiogenesis, leading to hyperproliferative diseases such as cancer,make the present invention particularly useful for interruptingHGF-mediated physiological events, for targeting substances, e.g.,therapeutics, including radiotherapeutics, to such sites, and forimaging important sites of cellular hyperproliferation.

In answer to the need for improved materials and methods for detecting,localizing, imaging, measuring and possibly inhibiting or affecting,e.g., hyperproliferation and/or angiogenesis, it has been surprisinglydiscovered that twelve classes of non-naturally occurring polypeptidesbind specifically to cMet. Appropriate labeling of such polypeptidesprovides detectable imaging agents that can bind, e.g., at highconcentration, to cMet-expressing cells or cells exhibiting HGF/cMetcomplexes, providing specific imaging agents for sites of cellularproliferation and/or angiogenesis. The cMet binding polypeptides of theinstant invention can thus be used in the detection and diagnosis ofsuch hyperproliferative-related and/or angiogenesis-related disorders.Conjugation or fusion of such polypeptides with effective agents such ascMet inhibitors or tumoricidal agents also can be used to treatpathogenic tumors, e.g., by causing the conjugate or fusion to “home” tothe site of active proliferation and/or angiogenesis, thereby providingan effective means for treating pathogenic conditions associated withhyperproliferation and/or angiogenesis.

This invention pertains to cMet binding polypeptides, and includes useof a single binding polypeptide as a monomer or in a multimeric orpolymeric construct as well as use of more than one binding polypeptideof the invention in multimeric or polymeric constructs. Bindingpolypeptides according to this invention are useful in any applicationwhere binding, inhibiting, detecting or isolating cMet, or fragmentsthereof retaining the polypeptide binding site, is advantageous. Aparticularly important aspect of such binding polypeptides is theinhibition of cMet activity, either through competition with HGF forcMet binding, or by directly inhibiting cMet activity irrespective ofwhether HGF is bound or not. For example, in some cases, cMet signalingcan occur in the absence of HGF binding, in such situations, a bindingpolypeptide that inhibits cMet signaling activity irrespective ofwhether HGF is bound, would be useful in inhibiting cMet signaling.

Another particularly advantageous use of the binding polypeptidesdisclosed herein is in a method of imaging cellular proliferation and/orangiogenesis in vivo. The method entails the use of specific bindingpolypeptides according to the invention for detecting a site of cellularproliferation and/or angiogenesis, where the binding polypeptides havebeen detectably labeled for use as imaging agents, including magneticresonance imaging (MRI) contrast agents, x-ray imaging agents,radiopharmaceutical imaging agents, ultrasound imaging agents, andoptical imaging agents.

Yet another advantageous use of the cMet binding polypeptides disclosedherein is to target therapeutic agents, (including compounds capable ofproviding a therapeutic, radiotherapeutic or cytotoxic effect) ordelivery vehicles for therapeutics (including drugs, genetic material,etc.) to sites of hyperproliferation and/or angiogenesis or other tissueexpressing cMet.

The cMet receptor is part of the receptor tyrosine kinase family ofsignaling molecules. For the purposes of the present invention, receptortyrosine kinase function can include any one of: oligomerization of thereceptor, receptor phosphorylation, kinase activity of the receptor,recruitment of downstream signaling molecules, induction of genes,induction of cell proliferation, induction of cell migration, orcombination thereof. “Heteromeric” molecules, used herein to refer tomolecules containing more than one cMet binding peptide as describedherein, such that each binding peptide of the heteromeric molecule bindsto a different site, e.g., “epitope”, of cMet, also are encompassed bythe present invention. For example, heteromeric constructs of bindingpolypeptides provided herein could, for example, bind, via one bindingpeptide, to, for example, the HGF binding site of cMet, while anotherbinding peptide of the heteromeric molecule binds to a different highaffinity binding site of cMet. Targeting two or more distinct epitopeson cMet with a single binding construct can greatly improve the abilityof the construct to inhibit HGF binding and/or receptor function (suchinhibition can occur by direct inhibition of cMet irrespective of HGFbinding). Even binding peptides with weak ability to block receptoractivity can be used to generate heteromeric constructs having improvedability to block HGF-dependent and HGF-independent receptor function.

Therefore, the present invention is drawn to constructs comprising meansfor producing multimeric molecules comprising two or more bindingpolypeptides, at least one of which binds cMet. In one embodiment, themultimeric constructs comprise two or more copies of a single bindingpolypeptide or nucleotide sequence that encode two or more copies of asingle binding polypeptide. In another embodiment, the multimericconstructs of the present invention comprise two or more bindingpolypeptides or nucleotide sequence that encode two or more bindingpolypeptides, such that at least two of the binding polypeptides in theconstruct are specific for different epitopes of cMet. These constructsalso are referred to herein as “heteromeric constructs”,“heteromultimers”, etc. The constructs of the present invention also caninclude unrelated, or control peptide. The constructs can include two ormore, three or more, or four or more binding polypeptides or thenucleotide sequences that encode such polypeptides. Based on theteachings provided herein, one of ordinary skill in the art is able toassemble the binding polypeptides provided herein into multimericconstructs and to select multimeric constructs having improvedproperties, such as improved ability to bind the target molecule, orimproved ability to inhibit receptor tyrosine kinase function. Suchmultimeric constructs having improved properties are included in thepresent invention.

Consensus sequences from the screen of the cyclic/linear peptidelibraries have been determined based on the twelve classes of specificcMet binding polypeptides shown in Table 6. In specific embodiments,cMet binding polypeptides of the invention comprise one or more of thesesequences. Such preferred cMet binding polypeptides include polypeptideswith the potential to form a cyclic or loop structure between invariantcysteine residues comprising.

The polypeptides described herein can have additional amino acidsattached at either or both of the - and C-terminal ends. In preferredembodiments, binding polypeptides according to the invention can beprepared having N-terminal and/or C-terminal flanking peptides of one ormore, preferably two, amino acids corresponding to the flanking peptidesof the display construct of the phage selectant from which the bindingpolypeptides were isolated. Preferred N-terminal flanking peptidesinclude Gly-Ser- (most preferably for TN6 sequences), Ala-Gly- (mostpreferably for TN8 and TN9 sequences), Gly-Ser- (most preferably forTN10 and TN11 sequences), Gly-Asp-(most preferably for TN12 sequences),Ala-Gln- (most preferably for linear sequences). Preferred C-terminalflanking peptides include -Ala-Pro (most preferably for TN6 sequences),-Gly-Thr (most preferably for TN8 and TN9 sequences), -Ala-Pro (mostpreferably for TN10 and TN11 sequences), -Asp-Pro (most preferably forTN12 sequences), -Asp-Phe (most preferably for linear sequences). Singleterminal amino acids also can be added to the binding polypeptides ofthe invention, and preferred terminal amino acids will correspond to theparental phage display construct, e.g., most preferably, N-terminalamino acids will be selected from Gly- (most preferably for TN6, TN8 andTN9 sequences), Ser- (most preferably for TN10 and TN11 sequences), Asp-(most preferably for TN12 sequences), and Gln- (most preferably forlinear sequences), and most preferably C-terminal amino acids will beselected from -Gly (most preferably for TN6, TN8 and TN9, and linearsequences), -Ala (most preferably for TN10 and TN11 sequences), and -Asp(most preferably for TN12 sequences). Conservative substitutions (i.e.,substitute amino acids selected within the following groups: {Arg, His,Lys}, {Glu, Asp}, {Asn, Cys, Glu, Gly, Ser, Thr, Tyr}, {Ala, Ile, Leu,Met, Phe, Pro, Trp, Val}) for such flanking amino acids also arecontemplated.

Examination of the sequence information and binding data from theisolates of libraries containing polypeptides with the potential to formloop structures (e.g., libraries designated TN6, TN8, TN9, TN10, TN11and TN12; the number refers to the number of amino acids in the sequencefrom cysteine to cysteine; additionally, the linear display library,LN20, also was screened) identifies an additional series of cMet bindingpolypeptides. A consensus motif was obtained from this initial screen ofa TN9 library (CxGpPxFxC; SEQ ID NO:512). The consensus sequence wasderived from the sequences listed in Table 6. This consensus sequencealong with sequence trends in the cMet binding peptides identified fromthe linear peptide library was used to design a second generationlibrary that was used in a secondary screen. Sequences from both screenswere used to identify twelve classes of cMet binding motifs listed inTable 6.

Another aspect of the present invention relates to modifications of thepolypeptides of the invention to provide specific cellular proliferationand/or angiogenesis imaging agents by detectably labeling a polypeptideor multimeric polypeptide construct according to the present invention.Such detectable labeling can involve radiolabeling, enzymatic labeling,or labeling with MR paramagnetic chelates or microparticles;incorporation into ultrasound bubbles, microparticles, microspheres,emulsions, or liposomes; or conjugation with optical dyes.

In another aspect of the present invention, methods for isolatingcMet-expressing cells using the present binding polypeptides ormultimeric polypeptide construct are provided.

Additionally, the cMet binding polypeptides or multimeric polypeptideconstruct of the invention can be used as therapeutic agents, eitheralone in a pharmaceutically acceptable composition or conjugated to (orin combination with) other therapeutic agents. The compositions can beused to treat diseases or conditions involving cellular proliferation,angiogenesis and/or wound healing.

When used as therapeutic agents, it may be advantageous to enhance theserum residence time of the peptides. This can be accomplished by: a)conjugating to the peptide a moiety, such as maleimide, that reacts withfree sulfhydryl groups on serum proteins, such as serum albumin, b)conjugating to the peptide a moiety, such as a fatty acid, that bindsnon-covalently to serum proteins, especially serum albumin, c)conjugating to the peptide a polymer, such as polyethylene glycol (PEG),that is known to enhance serum residence time, and d) fusing DNA thatencodes the cMet-binding peptide to DNA that encodes a serum proteinsuch as human serum albumin or an antibody and expressing the encodedfusion protein.

In another aspect of the invention, methods of screening polypeptidesidentified by phage display for their ability to bind to cellsexpressing the target are provided. These methods permit rapid screeningof the binding ability of polypeptides, including polypeptides withmonomeric affinities that are too low for evaluation in standardcell-binding assays. Additionally, these methods can be used to rapidlyassess the stability of the peptides in the presence of serum.

In one embodiment, the present invention is directed to a polypeptide ormultimeric polypeptide construct having the ability to bind to cMet or acomplex comprising cMet and HGF comprising an amino acid sequencecomprising Cys-X₁-Gly-X₂-Pro-X₃-Phe-X₄-Cys, wherein X₁, X₂, X₃ and X₄can be any amino acid. In a particular embodiment, X₂ is Pro.

In another embodiment, the polypeptides of the invention furthercomprises N-terminal and/or C-terminal flanking peptides of one or moreamino acids. For example, the polypeptide can comprise a modificationselected from the group consisting of an amino acid substitution, andamide bond substitution, a D-amino acid substitution, a glycosylatedamino acid, a disulfide mimetic substitution, an amino acidtranslocation, a retro-inverso peptide, a peptoid, a retro-inversopeptoid and a synthetic peptide. In another embodiment, any of thepolypeptides described herein can be conjugated to a detectable label ora therapeutic agent, optionally further comprising a linker or spacerbetween the polypeptide and the detectable label or the therapeuticagent. In a particular embodiment, the detectable label or thetherapeutic agent is selected from the group consisting of: an enzyme, afluorescent compound, a liposome, an optical dye, a paramagnetic metalion, an ultrasound contrast agent and a radionuclide. In a particularembodiment, the therapeutic agent or detectable label comprises aradionuclide. For example, the radionuclide can be one or more selectedfrom the group consisting of ¹⁸F, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹²³I, ⁷⁷Br, ⁷⁶Br,^(99m)Tc, ⁵¹Cr, ⁶⁷Ga, ⁶⁸Ga, ⁴⁷Sc, ⁵¹Cr, ¹⁶⁷Tm, ¹⁴¹Ce, ¹¹¹In, ¹⁶⁸Yb,¹⁷⁵Yb, ¹⁴⁰La, ⁹⁰Y, ⁸⁸Y, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁶⁵Dy, ¹⁶⁶Dy, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu,⁹⁷Ru, ¹⁰³Ru, ¹⁸⁶Re, ¹⁸⁸Re, ²⁰³Pb, ²¹¹Bi, ²¹²Bi, ²¹³Bi, ²¹⁴Bi, ¹⁰⁵Rh,¹⁰⁹Pd, ^(117m)Sn, ¹⁴⁹Pm, ¹⁶¹Tb, ¹⁷⁷Lu, ¹⁹⁸Au and ¹⁹⁹Au. In anotherembodiment, the therapeutic agent or detectable label further comprisesa chelator. For example, the chelator can comprise a compound selectedfrom the group consisting of formula 20, 21, 22, 23a, 23b, 24a, 24b and25. In a particular embodiment, the radionuclide is ^(99m)Tc or ¹¹¹In.In another embodiment, the radionuclide is selected from the groupconsisting of ¹⁷⁷Lu, ⁹⁰Y, ¹⁵³Sm and ¹⁶⁶Ho. In another embodiment, thedetectable label comprises an ultrasound contrast agent. For example,the ultrasound contrast agent can comprise a phospholipid stabilizedmicrobubble or a microballoon comprising a gas, e.g., a fluorinated gas.In another embodiment, the detectable label comprises a paramagneticmetal ion and a chelator. Another aspect of the invention is directed toany of the polypeptides of the invention, wherein the therapeutic agentis selected from the group consisting of: a bioactive agent, a cytotoxicagent, a drug, a chemotherapeutic agent or a radiotherapeutic agent. Inother embodiments, the polypeptide has an apparent K_(D) for cMet ofcMet/HGF complex of less than about 10 μM, less than about 1.0 μM, lessthan about 0.1 μM or less than about 1 nM.

In one embodiment, the present invention is directed to a polypeptide ormultimeric polypeptide construct having the ability to bind to cMet or acomplex comprising cMet and HGF comprising an amino acid sequence of oneof the following classes: Class 1:X₁-X₂-X₃-Cys-X₄-X₅-X₆-X₇-Cys-X₈-X₉-X₁₀ (TN46), wherein X₁ is Phe, Leu,Ser, Trp, Tyr or Met; X₂ is Ile, Tyr, His, Thr or Asn; X₃ is Ile, Leu,Asp, Met, Phe or Ser; X₄ is Arg, Asn, Glu, Pro or Trp; X₅ is Glu, Gly,Leu, Pro, Thr, Trp or Tyr; X₆ is Asp, Gln, Glu Gly, Phe, Ser, Thr orTrp; X₇ is Ala, Arg, Asn, Gln, Glu, Gly, Phe, or Trp; X₈ is Gly, Asn,His, Arg, Met, Ile, Asp, Val or Thr; X₉ is Ser, Lys, Phe, Met, Thr, Aspor Leu; and X₁₀ is Ser, Pro, Thr, Leu, Tyr, Asn, His, Glu or Trp; orClass II: X₁-X₂-X₃-Cys-X₄-X₅-X₆-X₇-X₈-X₉-Cys-X₁₀-X₁₁-X₁₂ (TN8), whereinX₁ is Gly, Val, Trp, Thr, Lys or Gln; X₂ is Trp, Tyr, Leu, Phe or Thr;X₃ is Trp, Glu, Phe, Ile, Leu and Ser; X₄ is Asn, Gln or Glu; X₅ is Leu,Glu or Trp; X₆ is Glu, Ser or Tyr; X₇ is Glu, Met or Pro; X₈ is Met, Seror Trp; X₉ is Leu, Phe or Val; X₁₀ is Asp, Glu or Trp; X₁₁ is Met, Pheor Trp; and X₁₂ is Gln, Leu or Trp; or Class III:X₁-X₂-X₃-Cys-X₄-Gly-X₅-Pro-X₆-Phe-X₇-Cys-X₈-X₉ (TN9), wherein X₁ is Glu,Ser, Trp or Tyr; X₂ is Phe, Thr or Trp; X₃ is His, Phe or Trp; X₁ isAla, Lys, Ser or Thr; X₅ is Pro or Trp; X₆ is Ser or Thr; X₇ is Glu orSer; X₈ is Ile, Tip or Tyr; and X₉ is Glu, Met, Tip or Tyr; or ClassIV-1: X₁-X₂-X₃-Cys-X₄-Gly-Pro-Pro-X₅-Phe-X₆-Cys-Trp-X₇-X₈-X₉-X₁₀-X₁₁(TN9), wherein X₁ is Arg, Asp, Asn, Ile or Ser; X₂ is Leu, Ile, Phe, Tipor Val; X₃ is Asn, Gln, His, Leu, Tyr or Val; X₄ is Leu, Lys or Ser; X₅is Ala, Ser, Thr or Trp; X₆ is Leu, Ser or Tip; X₇ is Leu, Ser or Trp;X₈ is Phe or Tyr; X₉ is Asp, Glu, Gly or Val; X₁₀ is Met, Pro, Thr orSer; and X₁₁ is Glu or Gly; or Class IV-2:X₁-X₂-X₃-X₄-Trp-X₅-Cys-X₆-Gly-Pro-Pro-Thr-Phe-Glu-Cys-Trp-X₇-X₈ (TN9),wherein X₁ is Asp, Glu or Val; X₂ is Ala, Asp, Gly, Ser or Val; X₃ isAsp, Gly, Ser or Val; X₄ is Arg, Asn, Gly, Ser or Thr; X₅ is Gln or His;X₆ is Asn, Lys or Ser; X₇ is Ser or Tip; and X₈ is Phe or Tyr; or ClassV: X₁-X₂-X₃-Cys-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-Cys-X₁₂-X₁₃-X₁₄ (TN10),wherein X₁ is His, Phe, Pro, Thr or Trp; X₂ is Ala, Arg, Glu, His, Lysor Phe; X₃ is Met, Phe, Pro, Thr or Val; X₄ is His, Leu, Met, Phe orTrp; X₅ is Arg, Asp, Glu, Gly, Met or Trp; X₆ is Glu, Gly, Ile, Lys, Pheor Pro; X₇ is Asp, Phe, Pro, Ser, Trp or Tyr; Xg is Ala, Arg, Asn, Pheor Ser; X₉ is Ala, Gln, Gly, Leu or Phe; X₁₀ is Gln, Gly, Ile, Leu, Tipor Tyr; X₁₁ is Arg, Asp, Phe, Pro, Tyr or Val; X₁₂ is Asn, Gln, His, Ileor Thr; X₁₃ is Ala, Asn, Asp, Glu or His; and X₁₄ is Asn, Gln, Glu, Hisor Val; or Class VI:X₁-X₂-X₃-Cys-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-Cys-X₁₃-X₁₄-X₁₅, wherein X₁is Gln, Gly, Met, Phe or Ser; X₂ is Asn, Gln, Leu or Met; X₃ is Arg,Asn, Gly, His or Ile; X₄ is Asn, Asp, Leu, Thr or Tip; X₅ is Arg, Gln,Thr, Tyr or Val; X₆ is Glu, Gly, Leu, Met or Thr; X₇ is Ala, Asn, Asp,His, Ile, Leu or Ser; X₈ is Arg, Gln, Ser, Thr or Tyr; X₉ is Asp, Gly,Ile or Phe; X₁₀ is Gln, Phe or Thr; X₁₁ is Gln, His, Phe, Pro, Ser orTyr; X₁₂ is Asn, Asp, Phe, Pro or Ser; X₁₃ is Ala, Asn, Gly, Leu or Ser;X₁₄ is Arg, Pro, Ser or Val; and X₁₅ is Asp, Glu, Leu or Met; or ClassVIII: X₁-X₂-X₃-Cys-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-Cys-X₁₄-X₁₅-X₁₆,wherein X₁ is Ala, His, Leu, Phe or Tyr; X₂ is Arg, Asp, Leu, Ser orTyr; X₃ is Glu, Met or Tip; X₄ is Asp, Gln, Glu, Phe or Ser; X₅ is Glu,Ile, Phe or Tip; X₆ is Asn, Asp or Ser; X₇ is Asn, Asp or Leu; X₈ isAsp, Glue or Lys; X₉ is Gly, Phe or Thr; X₁₀ is Gly, Phe, Tip or Tyr;X₁₁ is Glu, Ser or Tip; X₁₂ is Glu, Phe, Tyr or Val; X₁₃ is Glu, Lys,Thr or Val; X₁₄ is Glu or Tip; X₁₅ is Asp, Phe, Pro, Ser or Trp; and X₁₆is Ala, Asn or Ile; or Class IX-1:Ser-Cys-X₁-Cys-X₂-Gly-Pro-Pro-Thr-Phe-Glu-Cys-Trp-Cys-Tyr-X₃-X₄-X₅,wherein X₁ is Asn, His or Tyr; X₂ is Gly or Ser; X₃ is Ala, Asp, Glu,Gly or Ser; X₄ is Ser or Thr; and X₅ is Asp or Glu; or Class IX-2:Glu-X₁-Gly-Ser-Cys-His-Cys-Ser-Gly-Pro-Pro-Thr-Phe-Glu-Cys-X₂-Cys-X₃,wherein X₁ is Ala, Glu, Gly or Ser; X₂ is Phe, Trp or Tyr; and X₃ is Pheor Tyr.

In another embodiment, the invention is directed to a polypeptide ormultimeric polypeptide construct having the ability to bind to cMet or acomplex comprising cMet and HGF comprising an amino acid sequence,wherein the amino acid sequence comprises at least six amino acids outof a contiguous stretch of nine amino acids from a sequence selectedfrom the group consisting of SEQ ID NOS:1-511. In a particularembodiment, the polypeptide, used as either a monomer or in a multimericconstruct, can be selected from the group consisting of SEQ IDNOS:1-511, SEQ ID NOS:1-10, SEQ ID NOS:11-47, SEQ ID NOS:48-101, SEQ IDNOS:102-364, SEQ ID NOS:365-370, SEQ ID NOS:371-387, SEQ ID NO:388 orSEQ ID NO:399, SEQ ID NOS:390-404, SEQ ID NOS:405-447, SEQ ID NO:448,SEQ ID NOS:449-496 and SEQ ID NOS:497-511.

In another embodiment, the invention is directed to a method forisolating phage that bind cMet or a complex comprising cMet and HGF,comprising the steps of: immobilizing cMet or a complex comprising cMetand HGF on a solid support; contacting a library of potential cMet orcMet/HGF complex binding phage with the solid support to bind cMet orcMet/HGF binding phage in the library; and removing the unbound portionof the phage library from the solid support, thereby isolating phagethat bind cMet or a complex comprising cMet and HGF.

In another embodiment, the invention is directed to a method ofdetecting cMet or a complex comprising cMet and HGF in an animal orhuman subject and optionally imaging at least a portion of the animal orhuman subject comprising the steps of: detectably labeling a polypeptideor multimeric polypeptide construct having the ability to bind to cMetor a complex comprising cMet and HGF comprising an amino acid sequencecomprising Cys-X₁-Gly-X₂-Pro-X₃-Phe-X₄-Cys, wherein X₁, X₂, X₃ and X₄can be any amino acid; administering to the subject the labeledpolypeptide or multimeric polypeptide construct; and, detecting thelabeled polypeptide or construct in the subject, and, optionally,constructing an image, thereby detecting cMet or a complex comprisingcMet and HGF.

In a particular embodiments, the methods of the invention encompassmethods wherein the label is selected from the group consisting of: anenzyme, a fluorescent compound, an ultrasound contrast agent, a liposomeand an optical dye, wherein the label optionally further comprises alinker and/or a spacer. In particular embodiment, the ultrasoundcontrast agent is a phospholipid stabilized microbubble or amicroballoon comprising a gas, e.g., a fluorinated gas. In otherembodiments, the label is a radioactive label or a paramagnetic metalatom, and optionally further comprises a linker or a spacer. In anotherembodiment, the radioactive label comprises a radionuclide selected fromthe group consisting of: ¹⁸F, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹²³I, ⁷⁷Br, ⁷⁶Br,^(99m)Tc, ⁵¹Cr, ⁶⁷Ga, ⁶⁸Ga, ⁴⁷Sc, ⁵¹Cr, ¹⁶⁷Tm, ¹⁴¹Ce, ¹¹¹In, ¹⁶⁸Yb,¹⁷⁵Yb, ¹⁴⁰La, ⁹⁰Y, ⁸⁸Y, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁶⁵Dy, ¹⁶⁶Dy, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu,⁹⁷Ru, ¹⁰³Ru, ¹⁸⁶Re, ¹⁸⁸Re, ²⁰³Pb, ²¹¹Bi, ²¹²Bi, ²¹³Bi, ²¹⁴Bi, ¹⁰⁵Rh,¹⁰⁹Pd, ^(117m)Sn, ¹⁴⁹Pm, ¹⁶¹Tb, ¹⁷⁷Lu, ¹⁹⁸Au and ¹⁹⁹Au. In anotherembodiment, the radioactive label further comprises a chelator, e.g.,chelators selected from the group consisting of: formula 20, 21, 22,23a, 23b, 24a, 24b and 25. In another embodiment, the radionuclide is^(99m)Tc or ¹¹¹In. In a particular embodiment, the paramagnetic labelcomprises a paramagnetic metal atom selected from the group consistingof: Mn²⁺, Cu²⁺, Fe²⁺, Co²⁺, Ni²⁺, Gd³⁺, Eu³⁺, Dy³⁺, Pr³⁺, Cr³⁺, Co³⁺,Fe³⁺, Ti³⁺, Tb³⁺, Nd³⁺, sm³⁺, Ho³⁺, Er³⁺, Pa⁴⁺ and Eu²⁺. In anotherembodiment, the paramagnetic label further comprises a chelator, e.g., achelator is selected from the group consisting of: DTPA, DO3A, DOTA,EDTA, TETA, EHPG, HEED, NOTA, DOTMA, TETMA, PDTA, TTHA, LICAM, andMECAM. In particular embodiments, detection of the labeled polypeptideor multimeric polypeptide construct is indicative of ahyperproliferative disorder. In other embodiments, detection of thelabeled polypeptide or multimeric polypeptide construct is indicative ofangiogenesis or neovascularization. In particular embodiments, the labelis an ultrasound contrast agent that comprises a fluorinated gasselected from the group of SF₆ freons, CF₄, C₂F₆, C₃F₈, C₄F_(10i) CBrF₃,CCl₂F₂, C₂CIF₅, CBrCIF₂ and perfluorocarbons. In particular embodiments,the ultrasound contrast agent comprises a perfluorocarbon gas having theformula C_(n)F_(n+2) wherein n is from 1 to 12.

In another embodiment, the invention is directed to a method ofdetecting cMet or a complex comprising cMet and HGF in an animal orhuman subject and optionally imaging at least a portion of the animal orhuman subject comprising the steps of: detectably labeling a polypeptideor multimeric polypeptide construct having the ability to bind to cMetor a complex comprising cMet and HGF comprising an amino acid sequence,wherein the amino acid sequence comprises at least six amino acids outof a contiguous stretch of nine amino acids from a sequence selectedfrom the group consisting of SEQ ID NOS:1-511; administering to thesubject the labeled polypeptide or construct; and, detecting the labeledpolypeptide or construct in the subject, and, optionally, constructingan image, thereby detecting cMet or a complex comprising cMet and HGF.

In another embodiment, the invention is directed to a method of treatinga condition involving activation of cMet, comprising administering to ananimal or human subject in need of treatment for such a condition acomposition comprising a polypeptide or multimeric polypeptide constructhaving the ability to bind to cMet or a complex comprising cMet and HGFcomprising an amino acid sequence comprisingCys-X₁-Gly-X₂-Pro-X₃-Phe-X₄-Cys, wherein X₁, X₂, X₃ and X₄ can be anyamino acid. In another embodiment, the invention is directed to a methodof treating a condition involving activation of cMet, comprisingadministering to an animal or human subject in need of treatment forsuch a condition a composition comprising a polypeptide or multimericpolypeptide construct having the ability to bind to cMet or a complexcomprising cMet and HGF comprising an amino acid sequence, wherein theamino acid sequence comprises at least six amino acids out of acontiguous stretch of nine amino acids from a sequence selected from thegroup consisting of SEQ ID NOS:1-511. In a particular embodiment, thecondition is solid tumor growth, e.g., wherein the tumor is selectedfrom the group consisting of breast, thyroid, glioblastoma, prostate,malignant mesothelioma, colorectal, hepatocellular, hepatobiliary,renal, osteosarcoma and cervical. In a particular embodiment, thepolypeptide or multimeric polypeptide construct can be conjugated to atumoricidal agent.

In another embodiment, the invention is directed to a recombinantbacteriophage displaying any one or more of the polypeptides ormultimeric polypeptide construct described herein or having any one ormore of the consensus sequences described herein, such that the phagehas the ability to bind to cMet or a complex comprising cMet and HGF,and wherein the polypeptide is displayed on the surface of therecombinant bacteriophage.

In another embodiment, the invention is directed to a magnetic resonanceimaging contrast agent comprising a composition comprising a polypeptidehaving the ability to bind to cMet or a complex comprising cMet and HGFcomprising an amino acid sequence comprisingCys-X₁-Gly-X₂-Pro-X₃-Phe-X₄-Cys, wherein X₁, X₂, X₃ and X₄ can be anyamino acid, or wherein the amino acid sequence comprises at least sixamino acids out of a contiguous stretch of nine amino acids from asequence selected from the group consisting of SEQ ID NOS:1-511. In aparticular embodiment, the magnetic resonance imaging contrast agentfurther comprises at least one paramagnetic metal atom, e.g., at leastone chelator selected from the group consisting of: DTPA, DOTA, EDTA,TETA, EHPG, HBED, NOTA, DOTMA, TETMA, PDTA, TTHA, LICAM, and MECAM. Inparticular embodiments, the chelator is selected from the groupconsisting of: diethylenetriamine, tetraazacyclododecane and acarboxymethyl-substituted derivative thereof. In other embodiments, theparamagnetic metal atom is selected from the group consisting of Mn²⁺,Cu²⁺, Fe²⁺, Co²⁺, Ni²⁺, Gd³⁺, Eu³⁺, Dy³⁺, Pr³⁺, Cr³⁺, Co³⁺, Fe³⁺, Ti³⁺,Tb³⁺, Nd³⁺, Sm³⁺, Ho³⁺, Er³⁺, Pa⁴⁺ and Eu²⁺. In a particular embodiment,the multivalent cation is Gd³⁺.

In another embodiment, the invention is directed to a method foridentifying cMet or cMet/HGF complex binding compounds comprising thesteps of: utilizing a cMet or cMet/HGF complex binding polypeptide ormultimeric polypeptide construct having the ability to bind to cMet or acomplex comprising cMet and HGF comprising an amino acid sequencecomprising Cys-X₁-Gly-X₂-Pro-X₃-Phe-X₄-Cys, wherein X₁, X₂, X₃ and X₄can be any amino acid, to form a complex with a cMet or cMet/HGF complextarget; contacting the complex with one or more potential cMet orcMet/HGF complex binding compounds; and determining whether thepotential cMet or cMet/HGF complex binding compound competes with thecMet or cMet/HGF complex binding polypeptide to form a complex with thecMet or cMet/HGF complex target.

In one embodiment, the invention is directed to a diagnostic imagingcontrast agent comprising a polypeptide or multimeric polypeptideconstruct having the ability to bind to cMet or a complex comprisingcMet and HGF comprising an amino acid sequence comprisingCys-X₁-Gly-X₂-Pro-X₃-Phe-X₄-Cys, wherein X₁, X₂, X₃ and X₄ can be anyamino acid, or wherein the amino acid sequence comprises at least sixamino acids out of a contiguous stretch of nine amino acids from asequence selected from the group consisting of SEQ ID NOS:1-511.

In another embodiment, the invention is directed to a method of medicalimaging comprising the steps of administering to an animal or humansubject a pharmaceutical preparation of a contrast agent comprising atleast one polypeptide or multimeric polypeptide construct having theability to bind to cMet or a complex comprising cMet and HGF comprisingan, amino acid sequence comprising Cys-X₁-Gly-X₂-Pro-X₃-Phe-X₄-Cys,wherein X₁, X₂, X₃ and X₄ can be any amino acid, and imaging thecontrast agent by a method selected from the group consisting of:magnetic resonance imaging, ultrasound imaging, optical imaging,sonoluminescence imaging, photoacoustic imaging, and nuclear imaging. Inanother embodiment, the amino acid sequence comprises at least six aminoacids out of a contiguous stretch of nine amino acids from a sequenceselected from the group consisting of SEQ ID NOS:1-511, and imaging thecontrast agent by a method selected from the group consisting of:magnetic resonance imaging, ultrasound imaging, optical imaging,sonoluminescence imaging, photoacoustic imaging, and nuclear imaging.

In another embodiment, the invention is directed to a method ofradiotherapy comprising administering to an animal or human subject inneed of such therapy a compound comprising at least one polypeptide ormultimeric polypeptide construct having the ability to bind to cMet or acomplex comprising cMet and HGF comprising an amino acid sequencecomprising Cys-X₁-Gly-X₂-Pro-X₃-Phe-X₄-Cys, wherein X₁, X₂, X₃ and X₄can be any amino acid, or wherein the amino acid sequence comprises atleast six amino acids out of a contiguous stretch of nine amino acidsfrom a sequence selected from the group consisting of SEQ ID NOS:1-511,conjugated to a radionuclide useful for radiotherapy. In a particularembodiment, the compound further comprises a chelator, e.g., a compoundselected from the group consisting of: formula 20, 21, 22, 23a, 23b,24a, 24b and 25. In another embodiment, the compound further comprises aspacer or linker. In a particular embodiment, the radionuclide can be¹⁸⁵Re, ¹⁸⁸Re, ¹⁷⁷Lu, ⁹⁰Y, ¹⁵³Sm or ¹⁶⁶Ho.

In another embodiment, the invention is directed to a kit forpreparation of a radiopharmaceutical comprising a polypeptide ormultimeric polypeptide construct having the ability to bind to cMet or acomplex comprising cMet and HGF comprising an amino acid sequencecomprising Cys-X₁-Gly-X₂-Pro-X₃-Phe-X₄-Cys, wherein X₁, X₂, X₃ and X₄can be any amino acid, or wherein the amino acid sequence comprises atleast six amino acids out of a contiguous stretch of nine amino acidsfrom a sequence selected from the group consisting of SEQ ID NOS:1-511,a chelator for a radionuclide, and a reducing agent.

In another embodiment, the invention is directed to a method oftargeting genetic material to cMet-expressing cells comprisingadministering to an animal or a human in need of such genetic material apolypeptide or multimeric polypeptide construct having the ability tobind to cMet or a complex comprising cMet and HGF comprising an aminoacid sequence comprising Cys-X₁-Gly-X₂-Pro-X₃-Phe-X₄-Cys, wherein X₁,X₂, X₃ and X₄ can be any amino acid, or wherein the amino acid sequencecomprises at least six amino acids out of a contiguous stretch of nineamino acids from a sequence selected from the group consisting of SEQ IDNOS:1-511, conjugated to or associated with the genetic material or adelivery vehicle containing such genetic material.

In another embodiment, the invention is directed to a method ofscreening binding polypeptides identified by phage display for theirability to bind to cells expressing the cMet or cMet/HGF targetcomprising the steps of preparing multimeric constructs including one ormore binding polypeptides; contacting the multimeric constructs withcells expressing the target and assessing the ability of the multimericconstructs to bind to the target. In a particular embodiment, the cellscan be engineered by recombinant DNA technology to express the target.In another embodiment, the multimeric constructs can be detectablylabeled. In another embodiment, the ability of the multimeric constructsto bind to the target is assessed in the presence of serum. In anotherembodiment, the multimeric construct can comprise biotinylated bindingpolypeptides complexed with avidin, streptavidin or neutravidin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C are representations of mimics, which can be employed tomimic structural motifs and turn features in a peptide andsimultaneously provide stability to proteolysis and enhance otherproperties (structure 1A: Hart, S, and Etzkorn, F., 1999. J. Org. Chem.,64:2998-2999; structure 1B: Hanessian, S, and McNaughton-Smith, G.,“Synthesis of a Versatile Peptidomimetic Scaffold” in Methods inMolecular Medicine, Vol. 23: Peptidomimetics Protocols, W. Kazmierski,Ed. (Humana Press Inc., Totowa, N.J., 1999), Chapter 10, pp. 161-174;structure 1C: WO 01/16135.

FIG. 2 is a representation of the amino acids (4), containing anaminoalcohol function, and (5) containing an alkoxyamino function.

FIG. 3 is a representation depicting the cyclization of Cysteine with apendant bromoacetamide function (this process is referred to herein as“scheme 1”).

FIG. 4 is a representation showing intramolecular cyclization ofsuitably located vicinal amino mercaptan functions and aldehydefunctions to provide thiazolidines that result in the formation of abicyclic peptide, one ring of which is that formed by the residues inthe main chain, and the second ring being the thiazolidine ring (thisprocess is referred to herein as “scheme 2”).

FIG. 5 is a representation showing how a lactam function, available byintramolecular coupling via standard peptide coupling reagents (such asHATU, PyBOP etc) can act as a surrogate for the disulfide bond. TheDde/Dmab approach is shown (and is referred to herein as “scheme 3”).

FIG. 6 is a representation showing the Grubbs reaction (referred toherein as “scheme 4”).

FIGS. 7A and 7B are chemical structures of phospholipid moieties.

FIGS. 8A-F depict structures of preferred metal chelators.

FIG. 9 is a schematic representation of the selection strategy that wasemployed to identify cMet binding polypeptides. TEA=triethylamine, BeadInfection=capture of non-eluted phage that remained bound to thecMet-Fc/protein-A beads.

FIG. 10 illustrates the growth inhibitory properties of cMet-bindingpeptide SEQ ID NO:365.

FIG. 11 shows a schematic diagram for the preparation of SEQ ID NO:514conjugated to a 6-PnAO-Glut moiety, (referred to herein as “scheme 5”).

FIG. 12 shows a schematic diagram for the preparation of a heterodimercontaining SEQ ID NOS: 514 and 515 joined by a K(PnAO6-Glut) linker(referred to herein as “scheme 5”).

FIGS. 13A-13C show the chemical structures of three heterodimers asfollows: FIG. 13A shows SEQ ID NO:514 linked to SEQ ID NO:515(Ac-GSPEMCMMFPFLYPCNHHAPGGGK{PnAO6-Glut-K[Ac-GSFFPCWRIDRFGYCHANAPGGGKJJ-Glut]-NH2}-NH2); FIG. 13Bshows SEQ ID NO:515 linked to SEQ ID NO:516 (Ac-GSFFPCWRIDRFGYCHANAPGGGK{PnAO6-Glut-K[Ac-AQEWEREYFVDGFWGSWFGIPHGGGK(JJ-Glut)-NH2]}-NH2); andFIG. 13C shows SEQ ID NO:514 linked to SEQ ID NO:517(Ac-GSPEMCMMFPFLYPCNIIHAPGGGK{PnAO6-Glut-K[Ac-GDYSECFFEPDSFEVKCYDRDPGGGK(JJ-Glut)-NH2]}-NH2).

FIG. 14 is a graphical representation of data showing binding ofderivatives of SEQ ID NO:514 with different spacer length and biotin.Derivatives have none, one J and two J spacers respectively in betweenthe targeting sequence and biotin.

DETAILED DESCRIPTION OF THE INVENTION

A description of preferred embodiments of the invention follows.

The present invention provides novel binding moieties that bind to thehepatocyte growth factor receptor (“HGFr” or “cMet”). Such bindingmoieties make possible the efficient detection, imaging and localizationof activated cells exhibiting upregulated cMet expression and binding ofHGF to cMet. Such activated cells are initiators of cellularproliferation, and therefore the polypeptides described herein provide ameans of detecting, monitoring and localizing sites of proliferation. Inparticular, the binding moieties of this invention, which includepolypeptides and multimeric polypeptide constructs, when appropriatelylabeled, are useful for detecting, imaging and localizing tumors orother proliferative disorders that result from dysregulated cellularproliferation (e.g., cancer). Thus, the binding polypeptides andmultimeric polypeptide constructs of the invention can be used to form avariety of diagnostic and therapeutic agents for diagnosing and treatingneoplastic tumor growth or other proliferative disorders. In addition,the binding polypeptides and multimeric polypeptide constructs canthemselves be used as therapeutic agents.

Specific cMet binding polypeptides according to the present inventionwere isolated initially by screening of phage display libraries, thatis, populations of recombinant bacteriophage transformed to express anexogenous peptide on their surface. In order to isolate new polypeptidebinding moieties for a particular target, such as cMet, screening oflarge peptide libraries, for example using phage display techniques, isespecially advantageous, in that very large numbers (e.g., 5×10⁹) ofpotential binders can be tested and successful binders isolated in ashort period of time.

In order to prepare a phage library of displaying polypeptides to screenfor binding polypeptides such as cMet binding polypeptides and/orpolypeptides that bind to a complex comprising HGF bound to cMet, acandidate binding domain is selected to serve as a structural templatefor the peptides to be displayed in the library. The phage library ismade up of a multiplicity of analogues of the parental domain ortemplate. The binding domain template can be a naturally occurring orsynthetic protein, or a region or domain of a protein. The bindingdomain template can be selected based on knowledge of a knowninteraction between the binding domain template and the binding target,but this is not critical. In fact, it is not essential for the selecteddomain to act as a template for the library or have any affinity for thetarget at all; its purpose is to provide a structure from which amultiplicity (library) of similarly structured polypeptides (analogues)can be generated, which multiplicity of analogs will include one or moreanalogs that exhibit the desired binding properties (and any otherproperties screened for).

In selecting the parental binding domain or template on which to basethe variegated amino acid sequences of the library, an importantconsideration is how the variegated peptide domains will be presented tothe target, i.e., in what conformation the peptide analogues will comeinto contact with the target. In phage display methodologies, forexample, the analogs are generated by insertion of synthetic DNAencoding the analogs into phage, resulting in display of the analog onthe surfaces of the phage. Such libraries of phage, such as M13 phage,displaying a wide variety of different polypeptides, can be preparedusing techniques as described, e.g., in Kay et al., Phage Display ofPeptides and Proteins: A Laboratory Manual (Academic Press, Inc., SanDiego, 1996) and U.S. Pat. No. 5,223,409 (Ladner et al.), incorporatedherein by reference.

In isolating the specific polypeptides according to this invention,seven cyclic peptide (or “loop”) libraries, designated TN6, TN7, TN8,TN9, TN10, TN11, TN12, and a linear library, designated LN20, wereinitially screened. Each library was constructed for expression ofdiversified polypeptides on M13 phage. The seven libraries having a “TN”designation were designed to display a short, variegated exogenouspeptide loop of 6, 7, 8, 9, 10, 11 or 12 amino acids, respectively, onthe surface of M13 phage, at the amino terminus of protein III. Thelibraries are designated TN6 (having a potential 3.3×10¹² amino acidsequence diversity), TN7 (having a potential 1.2×10¹⁴ amino acidsequence diversity), TN8 (having a potential 2.2×10¹⁵ amino acidsequence diversity), TN9 (having a potential 4.2×10¹⁶ amino acidsequence diversity, TN10 (having a potential 3.0×10¹⁶ amino acidsequence diversity), TN11 (having a potential 1.5×10¹⁹ amino acidsequence diversity), TN12 (having a sequence diversity of 4.6×10¹⁹), andLN20 (having a potential 3.8×10²⁵ amino acid sequence diversity).

The TN6 library was constructed to display a single microprotein bindingloop contained in a 12-amino acid template. The TN6 library utilized atemplate sequence of Xaa1-Xaa2-Xaa3-Cys-Xaa5-Xaa6Xaa7-Xaa8-Cys-Xaa10-Xaa11-Xaa12. The amino acids at positions 2, 3, 5,6, 7, 8, 10, and 11 of the template were varied to permit any amino acidexcept cysteine (Cys). The amino acids at positions 1 and 12 of thetemplate were varied to permit any amino acid except cysteine (Cys),glutamic acid (Glu), isoleucine (Ile), Lysine (Lys), methionine (Met),and threonine (Thr).

The TN7 library was constructed to display a single microprotein bindingloop contained in a 13-amino acid template. The TN7 library utilized atemplate sequence ofXaa1-Xaa2-Xaa3-Cys-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Cys-Xaa11-Xaa12-Xaa13. Theamino acids at amino acid positions 1, 2, 3, 5, 6, 7, 8, 9, 11, 12, and13 of the template were varied to permit any amino acid except cysteine(Cys).

The TN8 library was constructed to display a single microprotein bindingloop contained in a 14-amino acid template, The TN8 library utilized atemplate sequence ofXaa1-Xaa2-Xaa3-Cys-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Cys-Xaa12-Xaa13-Xaa14.The amino acids at position 1, 2, 3, 5, 6, 7, 8, 9, 10, 12, 13, and 14in the template were varied to permit any amino acid except cysteine(Cys).

The TN9 library was constructed to display a single microprotein bindingloop contained in a 15-amino acid template. The TN9 library utilized atemplate sequenceXaa1-Xaa2-Xaa3-Cys-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Xaa11-Cys-Xaa13-Xaa14-Xaa15.The amino acids at position 1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 13, 14 and15 in the template were varied to permit any amino acid except cysteine(Cys).

The TN10 library was constructed to display a single microproteinbinding loop contained in a 16-amino acid template. The TN10 libraryutilized a template sequence Xaa1Xaa2-Xaa3Cys-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Xaa11-Xaa12-Cys-Xaa14-Xaa15-Xaa16.The amino acids at positions 1, 2, 15, and 16 in the template werevaried to permit any amino acid selected from a group of 10 amino acids:D, F, H, L, N, P, R, S, W, or Y). The amino acids at positions 3 and 14in the template were varied to permit any amino acid selected from agroup of 14 amino acids: A, D, F, G, H, L, N, P, Q, R, S, V, W, or Y).The amino acids at positions 5, 6, 7, 8, 9, 10, 11, and 12 in thetemplate were varied to permit any amino acid except cysteine (Cys).

The TN11 library was constructed to display a single microproteinbinding loop contained in a 17-amino acid template. The TN11 libraryutilized a template sequenceXaa1-Xaa2-Xaa3-Cys-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Xaa11-Xaa12Xaa13-Cys-Xaa15-Xaa16-Xaa17. The amino acids at positions 1 through 3, 5through 13, and 15 through 17 in the template were varied to permit anyamino acid except cysteine (Cys).

The TN12 library was constructed to display a single microproteinbinding loop contained in an 18-amino acid template. The TN12 libraryutilized a template sequence Xaa1-Xaa2-Xaa3-Cys-Xaa5-Xaa6-Xaa7-Xaa8Xaa9-Xaa10-Xaa11-Xaa12-Xaa13-Xaa14-Cys-Xaa16-Xaa17-Xaa18. The aminoacids at position 1, 2, 17, and 18 in the template were varied to permitany amino acid selected from a group of 12 amino acids: A, D, F, G, H,L, N, P, R, S, W, or Y). The amino acids at positions 3, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, and 16 were varied to permit any amino acid exceptcysteine (Cys).

The LN20 library was constructed to display multiple linear peptides onthe surface of a phage. Each phage, however, displays multiple copies ofthe same sequence. Therefore, a single phage will display, for example,five copies of a particular sequence, a different phage will display,for example, five copies of a different sequence, etc. The linearpeptides are provided in a 20-amino acid template. The amino acids ateach position in the template were varied to permit any amino acidexcept cysteine (Cys).

The binding polypeptides provided herein can include additions ortruncations in the - and/or C-termini. Such modified bindingpolypeptides are expected to bind cMet. For example, a -GGGK linker (SEQID NO:513) can be present at the N-terminus of the binding polypeptidesprovided herein. Other linkers, such as -GSGK (SEQ ID NO:651), or -GSGSK(SEQ ID NO:652) could be used. Binding polypeptides comprising the loopportion of the templates and sequences provided herein are expected tobind cMet and also are encompassed by the present invention. The loopportion of the templates and sequences includes the sequences betweenand including the two cysteine residues that are expected to form adisulfide bond, thereby generating a peptide loop structure.Furthermore, the binding polypeptides of the present invention caninclude additional amino acid residues at the - and/or C-termini.

The phage display libraries were created by making a designed series ofmutations or variations within a coding sequence for the polypeptidetemplate, each mutant sequence encoding a peptide analog correspondingin overall structure to the template except having one or more aminoacid variations in the sequence of the template. The novel variegated(mutated) DNA provides sequence diversity, and each transformant phagedisplays one variant of the initial template amino acid sequence encodedby the DNA, leading to a phage population (library) displaying a vastnumber of different but structurally related amino acid sequences. Theamino acid variations are expected to alter the binding properties ofthe binding peptide or domain without significantly altering itsstructure, at least for most substitutions. It is preferred that theamino acid positions that are selected for variation (variable aminoacid positions) will be surface amino acid positions, that is, positionsin the amino acid sequence of the domains that, when the domain is inits most stable conformation, appear on the outer surface of the domain(i.e., the surface exposed to solution). Most preferably the amino acidpositions to be varied will be adjacent or close together, so as tomaximize the effect of substitutions.

As indicated previously, the techniques discussed in Kay et al., PhageDisplay of Peptides and Proteins: A Laboratory Manual (Academic Press,Inc., San Diego, 1996) and U.S. Pat. No. 5,223,409 are particularlyuseful in preparing a library of potential binders corresponding to theselected parental template. Libraries as discussed above were preparedaccording to such techniques, and they were screened for cMet bindingpolypeptides against an immobilized target, as explained in the examplesto follow.

In a typical screen, a phage library is contacted with and allowed tobind the target, or a particular subcomponent thereof. To facilitateseparation of binders and non-binders, it is convenient to immobilizethe target on a solid support. Phage bearing a target-binding moietyform a complex with the target on the solid support whereas non-bindingphage remain in solution and can be washed away with excess buffer.Bound phage are then liberated from the target by changing the buffer toan extreme pH (pH 2 or pH 10), changing the ionic strength of thebuffer, adding denaturants, or other known means. To isolate the bindingphage exhibiting the polypeptides of the present invention, a proteinelution is performed, i.e., some phage are eluted from the target usingHGF in solution (competitive elution). Additionally, for example, veryhigh affinity binding phage that could not be competed off during theovernight HGF incubation were captured by using the phage still bound tosubstrate for infection of E. coli cells.

The recovered phage can then be amplified through infection of bacterialcells and the screening process can be repeated with the new pool thatis now depleted in non-binders and enriched for binders. The recovery ofeven a few binding phage is sufficient to carry the process tocompletion. After a few rounds of selection, the gene sequences encodingthe binding moieties derived from selected phage clones in the bindingpool are determined by conventional methods, described below, revealingthe peptide sequence that imparts binding affinity of the phage to thetarget. When the selection process works, the sequence diversity of thepopulation falls with each round of selection until desirable bindersremain. The sequences converge on a small number of related binders,typically 10-50 out of about 10⁹ to 10¹⁰ original candidates from eachlibrary. An increase in the number of phage recovered at each round ofselection, and of course, the recovery of closely related sequences aregood indications that convergence of the library has occurred in ascreen. After a set of binding polypeptides is identified, the sequenceinformation can be used to design other secondary phage libraries,biased for members having additional desired properties.

Formation of the disulfide binding loop is advantageous because it leadsto increased affinity and specificity for such peptides. However, inserum, the disulfide bond can be opened by free cysteines or otherthiol-containing molecules. Thus, it could be useful to modify thecysteine residues to replace the disulfide cross-link with another lessreactive linkage. The —CH₂—S—S—CH₂— cross-link has a preferred geometryin which the dihedral bond between sulfurs is close to 90 degrees, butthe exact geometry is determined by the context of other side groups andthe binding state of the molecule. Preferred modifications of theclosing cross-link of the binding loop will preserve the overall bondlengths and angles as much as possible. Suitable such alternativecross—links include thioether linkages such as —CH₂—S—CH₂—CH₂—,—CH₂—CH₂—S—CH₂—, —CH₂—CH₂—S—CH₂—CH₂—; lactam or amide linkages such as—CH₂—NH—CO—CH₂— and —CH₂—CO—NH—CH₂—; ether linkages such as—CH₂—CH₂—O—CH₂—CH₂—; alkylene bridges such as —(CH₂)_(n)— (where n=4, 5,or 6); the linkage —CH₂—NH—CO—NH—CH₂—, and similar groups known in theart.

Although polypeptides containing a stable disulfide-linked binding loopare most preferred, linear polypeptides derived from the foregoingsequences can be readily prepared, e.g., by substitution of one or bothcysteine residues, which may retain at least some of the cMet bindingactivity of the original polypeptide containing the disulfide linkage.In making such substitutions for Cys, the amino acids Gly, Ser, and Alaare preferred, and it also is preferred to substitute both Cys residues,so as not to leave a single Cys that could cause the polypeptide todimerize or react with other free thiol groups in a solution. All suchlinearized derivatives that retain cMet binding properties are withinthe scope of this invention.

Direct synthesis of the polypeptides of the invention can beaccomplished using conventional techniques, including solid-phasepeptide synthesis, solution-phase synthesis, etc. Solid-phase synthesisis preferred (see, for example, Stewart et al., Solid-Phase PeptideSynthesis (W.H. Freeman Co., San Francisco, 1989); Merrifield, J., 1963,Am. Chem. Soc., 85:2149-2154; Bodanszky and Bodanszky, The Practice ofPeptide Synthesis (Springer-Verlag, New York, 1984)), incorporatedherein by reference.

Polypeptides according to the invention can also be preparedcommercially by companies providing peptide synthesis as a service(e.g., BACHEM Bioscience, Inc., King of Prussia, Pa.; Quality ControlledBiochemicals, Inc., Hopkinton, Mass.).

Automated peptide synthesis machines, such as manufactured byPerkin-Elmer Applied Biosystems, also are available.

The polypeptide compound is preferably purified after it has beenisolated or synthesized by either chemical or recombinant techniques.For purification purposes, there are many standard methods that may beemployed, including reversed-phase high pressure liquid chromatography(RP-HPLC) using an alkylated silica column such as C₄-, C₈- orC₁₈-silica. A gradient mobile phase of increasing organic content isgenerally used to achieve purification, for example, acetonitrile in anaqueous buffer, usually containing a small amount of trifluoroaceticacid. Ion-exchange chromatography can also be used to separate peptidesbased on their charge. The degree of purity of the polypeptide can bedetermined by various methods, including identification of a major largepeak on HPLC. A polypeptide that produces a single peak that is at least95% of the input material on an HPLC column is preferred. Even morepreferable is a polypeptide that produces a single peak that is at least97%, at least 98%, at least 99% or even 99.5% or more of the inputmaterial on an HPLC column.

To ensure that the peptide obtained using any of the techniquesdescribed above is the desired peptide for use in compositions of thepresent invention, analysis of the peptide composition can be carriedout. Such composition analysis can be conducted using high resolutionmass spectrometry to determine the molecular weight of the peptide.Alternatively, the amino acid content of the peptide can be confirmed byhydrolyzing the peptide in aqueous acid, and separating, identifying andquantifying the components of the mixture using HPLC, or an amino acidanalyzer. Protein sequenators, which sequentially degrade the peptideand identify the amino acids in order, can also be used to determine thesequence of the peptide.

cMet binding polypeptides according to the present invention also can beproduced using recombinant DNA techniques, utilizing nucleic acids(polynucleotides) encoding the polypeptides according to this inventionand then expressing them recombinantly, i.e., by manipulating host cellsby introduction of exogenous nucleic acid molecules in known ways tocause such host cells to produce the desired cMet binding polypeptides.Such procedures are within the capability of those skilled in the art(see, for example, Davis et al., Basic Methods in Molecular Biology(1986)), incorporated by reference. Recombinant production of shortpeptides, such as those described herein, might not be practical incomparison to direct synthesis, however recombinant means of productioncan be very advantageous where a cMet binding moiety of this inventionis incorporated in a hybrid polypeptide or fusion protein.

In the practice of the present invention, a determination of theaffinity of the cMet binding moiety for cMet relative to another proteinor target is a useful measure, and is referred to as specificity forcMet. Standard assays for quantitating binding and determining affinityinclude equilibrium dialysis, equilibrium binding, gel filtration, orthe monitoring of numerous spectroscopic changes (such as a change influorescence polarization) that result from the interaction of thebinding moiety and its target. These techniques measure theconcentration of bound and free ligand as a function of ligand (orprotein) concentration. The concentration of bound polypeptide ([Bound])is related to the concentration of free polypeptide ([Free]) and theconcentration of binding sites for the polypeptide, i.e., on cMet, (N),as described in the following equation:

[Bound]=N×[Free]/((1/K_(a))+[Free]).

A solution of the data to this equation yields the association constant,K_(a), a quantitative measure of the binding affinity. The associationconstant, Ic is the reciprocal of the dissociation constant, K_(D). TheK_(D) is more frequently reported in measurements of affinity. PreferredcMet binding polypeptides have a K_(D) for cMet in the range of, forexample, less than 1 nanomolar (nM), 1 nM to 100 micromolar (μM), whichincludes K_(D) values of less than 10 nM, less than 20 nM, less than 40nM, less than 60 nM, less than 80 nM, less than 1 μM, less than 5 μM,less than 10 μM, less than 20 μM, less than 40 μM, less than 60 μM, andless than 80 μM.

Where cMet binding moieties are employed as imaging agents, otheraspects of binding specificity become important; imaging agents operatein a dynamic system in that binding of the imaging agent to the target(cMet, e.g., on activated cells) might not be in a stable equilibriumstate throughout the imaging procedure. For example, when the imagingagent is initially injected, the concentration of imaging agent and ofagent-target complex rapidly increases. Shortly after injection,however, the circulating (free) imaging agent starts to clear throughthe kidneys or liver, and the plasma concentration of imaging agentbegins to drop. This drop in the concentration of free imaging agent inthe plasma eventually causes the agent-target complex to dissociate. Theusefulness of an imaging agent depends on the difference in rate ofagent-target dissociation relative to the clearing rate of the agent.Ideally, the dissociation rate will be slow compared to the clearingrate, resulting in a long imaging time during which there is a highconcentration of agent-target complex and a low concentration of freeimaging agent (background signal) in the plasma.

Quantitative measurement of dissociation rates can be performed usingseveral methods known in the art, such as fiber optic fluorimetry (see,for example, Anderson and Miller, 1988, Clin. Chem., 34:1417-21),surface plasmon resonance (see, for example, Malmborg et al., 1996, J.Immunol. Methods, 198:51-7; and Schuck, 1997, Curr. Op. Biotechnol.,8:498-502), resonant mirror, and grating coupled planar waveguiding(see, for example, Hutchinson, 1995, Molec. Biotechnol., 3:47-54).Automated biosensors are commercially available for measuring bindingkinetics: BIAcore surface plasmon resonance sensor (Biacore AB, UppsalaSE), IAsys resonant mirror sensor (Fisons Applied Sensor Technology,Cambridge GB), BIOS-1 grated coupled planar waveguiding sensor(Artificial Sensor Instruments, Zurich CH).

Methods of Screening Polypeptides Identified by Phage Display For TheirAbility To Bind To Cells Expressing The Target

In another aspect of the invention, methods of screening bindingpolypeptides identified by phage display for their ability to bind tocells expressing the target (and not to cells that do not express thetarget) are provided. These methods address a significant problemassociated with screening peptides identified by phage display:frequently the peptides so identified do not have sufficient affinityfor the target to be screened against target-expressing cells inconventional assays. However, ascertaining that a particularphage-identified peptide binds to cells that express the target (anddoes not bind to cells that do not) is a critical piece of informationin identifying binding peptides that are potential in vivo targetingmoieties, whether they are used as monomers or as part of a multimericconstruct. The method takes advantage of the increase in affinity andavidity associated with multivalent binding and permit screening ofpolypeptides with low affinities against target-expressing cells.

The method generally consists of preparation and screening of multimericconstructs including one or more binding polypeptides. For example,polypeptides identified by phage display as binding to a target arebiotinylated and complexed with avidin, streptavidin or neutravidin toform tetrameric constructs. These tetrameric constructs are thenincubated with cells that express the desired target and cells that donot, and binding of the tetrameric construct is detected. Binding can bedetected using any method of detection known in the art. For example, todetect binding the avidin, streptavidin, or neutravidin may beconjugated to a detectable marker (e.g., a radioactive label, afluorescent label, or an enzymatic label which undergoes a color change,such as HRP (horse radish peroxidase), TMB (tetramethyl benzidine) oralkaline phosphatase).

The biotinylated peptides are preferably complexed with neutravidin-HRP.Neutravidin exhibits lower non-specific binding to molecules than theother alternatives due to the absence of lectin binding carbohydratemoieties and cell adhesion receptor-binding RYD domain in neutravidin(Hiller, Y. et al., 1987. Biochem. J., 248:167-171; Alon, R. et al.,1990. Biochem. Biophys. Res. Commun., 170:1236-41).

The tetrameric constructs can be screened against cells that naturallyexpress the target or cells that have been engineered via recombinantDNA technologies to express the target (e.g., transfectants,transformants, etc.). If cells that have been transfected to express thetarget are used, mock transfected cells (i.e., cells transfected withoutthe genetic material encoding the target) can be used as a control.

The tetrameric complexes can optionally be screened in the presence ofserum. Thus, the assay also can be used to rapidly evaluate the effectof serum on the binding of peptides to the target.

The methods disclosed herein are particularly useful in preparing andevaluating combinations of distinct binding polypeptides for use indimeric or multimeric targeting constructs that contain two or morebinding polypeptides. Use of biotin/avidin complexes allows forrelatively easy preparation of tetrameric constructs containing one tofour different binding peptides. Furthermore, it has now been found thataffinity and avidity of a targeting construct can be increased byinclusion of two or more targeting moieties that bind to differentepitopes on the same target. The screening methods described herein areuseful in identifying combinations of binding polypeptides that couldhave increased affinity when included in such multimeric constructs.

In a preferred embodiment, the screening methods described herein can beused to screen cMet binding polypeptides identified by phage display,such as those described herein. These methods can be used to assess thespecific binding of cMet binding polypeptides to cells that express cMetor have been engineered to express cMet. Tetrameric complexes ofbiotinylated cMet binding polypeptides of the invention and, forexample, neutravidin-HRP can be prepared and screened against cellstransfected to express cMet as well as mock transfected cells, which donot express cMet.

The assay can be used to identify cMet binding polypeptides that bindspecifically to cMet-expressing cells (and do not bind to cells that donot express cMet) even when the monodentate K_(D) of the polypeptide ison the order of 200 nM-300 nM. The assay can be used to screenhomotetrameric constructs containing four copies of a single cMetbinding polypeptide of the invention as well as heterotetrameric(constructs containing two or more different cMet binding polypeptides).The methods described herein are particularly useful for assessingcombinations of cMet binding polypeptides for use in multimericconstructs, particularly constructs containing two or more cMet bindingpolypeptides that bind to different epitopes of cMet.

The assay also can be used to assess the effect of serum on the cMetbinding polypeptides.

Modification or Optimization of cMet Binding Polypeptides

As discussed, modification or optimization of cMet binding polypeptidesis within the scope of the invention and the modified or optimizedpolypeptides are included within the definition of “cMet bindingpolypeptides”. Specifically, a polypeptide sequence identified by phagedisplay can be modified to optimize its potency, pharmacokineticbehavior, stability and/or other biological, physical and chemicalproperties.

Substitution of Amino Acid Residues

For example, one can make the following isosteric and/or conservativeamino acid changes in the parent polypeptide sequence with theexpectation that the resulting polypeptides would have a similar orimproved profile of the properties described above:

Substitution of alkyl-substituted hydrophobic amino acids: includingalanine, leucine, isoleucine, valine, norleucine, S-2-aminobutyric acid,S-cyclohexylalanine or other simple alpha-amino acids substituted by analiphatic side chain from C1-10 carbons including branched, cyclic andstraight chain alkyl, alkenyl or alkynyl substitutions.

Substitution of aromatic-substituted hydrophobic amino acids: includingphenylalanine, tryptophan, tyrosine, biphenylalanine, 1-naphthylalanine,2-naphthylalanine, 2-benzothienylalanine, 3-benzothienylalanine,histidine, amino, alkylamino, dialkylamino, aza, halogenated (fluoro,chloro, bromo, or iodo) or alkoxy (from C₁-C₄)-substituted forms of theprevious listed aromatic amino acids, illustrative examples of whichare: 2-,3- or 4-aminophenylalanine, 2-,3- or 4-chlorophenylalanine,2-,3- or 4-methylphenylalanine, 2-,3- or 4-methoxyphenylalanine,5-amino-, 5-chloro-, 5-methyl- or 5-methoxytryptophan, 2′-, 3′-, or4′-amino-, 2′-, 3′-, or 4′-chloro-, 2,3, or 4-biphenylalanine, 2′,-3′, - or 4′-methyl-2, 3 or 4-biphenylalanine, and 2- or3-pyridylalanine.

Substitution of amino acids containing basic functions: includingarginine, lysine, histidine, ornithine, 2,3-diaminopropionic acid,homoarginine, alkyl, alkenyl, or aryl-substituted (from C₁-C₁₀ branched,linear, or cyclic) derivatives of the previous amino acids, whether thesubstituent is on the heteroatoms (such as the alpha nitrogen, or thedistal nitrogen or nitrogens, or on the alpha carbon, in the pro-Rposition for example. Compounds that serve as illustrative examplesinclude: N-epsilon-isopropyl-lysine, 3-(4-tetrahydropyridyl)-glycine,3-(4-tetrahydropyridyl)-alanine, N,N-gamma, gamma'-diethyl-homoarginine.Included also are compounds such as alpha methyl arginine, alpha methyl2,3-diaminopropionic acid, alpha methyl histidine, alpha methylornithine where alkyl group occupies the pro-R position of the alphacarbon. Also included are the amides formed from alkyl, aromatic,heteroaromatic (where the heteroaromatic group has one or morenitrogens, oxygens or sulfur atoms singly or in combination) carboxylicacids or any of the many well-known activated derivatives such as acidchlorides, active esters, active azolides and related derivatives) andlysine, ornithine, or 2,3-diaminopropionic acid.

Substitution of acidic amino acids: including aspartic acid, glutamicacid, homoglutamic acid, tyrosine, alkyl, aryl, arylalkyl, andheteroaryl sulfonamides of 2,4-diaminopriopionic acid, ornithine orlysine and tetrazole-substituted alkyl amino acids.

Substitution of side chain amide residues: including asparagine,glutamine, and alkyl or aromatic substituted derivatives of asparagineor glutamine.

Substitution of hydroxyl containing amino acids: including serine,threonine, homoserine, 2,3-diaminopropionic acid, and alkyl or aromaticsubstituted derivatives of serine or threonine. It is also understoodthat the amino acids within each of the categories listed above can besubstituted for another of the same group.

Substitution of Amide Bonds

Another type of modification within the scope of the invention is tosubstitute the amide bonds within the backbone of the polypeptide. Forexample, to reduce or eliminate undesired proteolysis, or otherdegradation pathways that diminish serum stability, resulting in reducedor abolished bioactivity, or to restrict or increase conformationalflexibility, one can substitute amide bonds within the backbone of thepeptides with functionality that mimics the existing conformation oralters the conformation in the manner desired. Such modifications canproduce increased binding affinity or improved pharmacokinetic behavior.It is understood that those knowledgeable in the art of peptidesynthesis can make the following amide bond changes for any amide bondconnecting two amino acids with the expectation that the resultingpeptides could have the same or improved activity: insertion ofalpha-N-methylamides or peptide amide backbone thioamides, removal ofthe carbonyl to produce the cognate secondary amines, replacement of oneamino acid with an aza-amino acid to produce semicarbazone derivatives,and use of E-olefins and substituted E-olefins as amide bond surrogates.

Introduction of D-Amino Acids

Another approach within the scope of the invention is the introductionof D-alanine, or another D-amino acid, distal or proximal to the labilepeptide bond. In this case it is also understood to those skilled in theart that such D-amino acid substitutions can, and at times, must bemade, with D-amino acids whose side chains are not conservativereplacements for those of the L-amino acid being replaced. This isbecause of the difference in chirality and hence side-chain orientation,which could result in the accessing of a previously unexplored region ofthe binding site of the target that has moieties of different charge,hydrophobicity, steric requirements etc. than that serviced by the sidechain of the replaced L-amino acid.

Modifications To Improve Pharmacokinetic or Pharmacodynamic Properties

It also is understood that use of one or more cMet binding polypeptidesin a particular application could be benefitted by modifications of thepeptide or formulations of the peptide to improve pharmacokinetic andpharmacodynamic behavior. It is expected that the properties of thepeptide can be changed by attachment of moieties anticipated to bringabout the desired physical or chemical properties. Such moieties can beappended to the peptide using acids or amines, via amide bonds or ureabonds, respectively, to the - or C-terminus of the peptide, or to thependant amino group of a suitably located lysine or lysine derivative,2,3-diaminopropionic acid, ornithine, or other amino acid in the peptidethat possesses a pendant amine group or a pendant alkoxyamine orhydrazine group. Conversely acidic amino acid side-chains such as thoseof Asp or Glu can be selectively unmasked and amidated with aminesbearing the desired modifying functionality, or they can be modified inthis manner before incorporation into the peptide chain. The moietiesintroduced can be groups that are hydrophilic, basic, or nonpolar alkylor aromatic groups depending on the peptide of interest and the extantrequirements for modification of its properties.

Glycosylation of Amino Acid Residues

Yet another modification within the scope of the invention isglycosylation of one or more amino acid residues (e.g., serine orthreonine residues) in the cMet binding polypeptide. Glycosylation,which can be carried out using standard conditions, can be used toenhance solubility, alter pharmacokinetics and pharmacodynamics or toenhance binding via a specific or non-specific interaction involving theglycosidic moiety.

Formation of Salts

It also is within the scope of the invention to form different saltsthat could increase or decrease the water solubility or the ease offormulation of these peptides. These may include, but are not restrictedto, N-methylglucamine (meglumine), acetate, oxalates, ascorbates, etc.

Structural Modifications which Retain Structural Features

Yet another modification within the scope of the invention is truncationof cyclic polypeptides. The cyclic nature of many polypeptides of theinvention limits the conformational space available to the peptidesequence, particularly within the cycle. Therefore truncation of thepeptide by one or more residues distal or even proximal to the cycle, ateither the N-terminal or C-terminal region could provide truncatedpeptides with similar or improved biological activity. A unique sequenceof amino acids, even as small as three amino acids, which is responsiblefor the binding activity, can be identified, as noted for RGD peptides(Plow, E. et al., 1987. Blood, 70:110-5; Oldberg, A. et al., 1988. J.Biol. Chem., 263:19433-19436; Taub, R. et al., 1989. J. Biol. Chem.,264:259-65; Andrieux, A. et al., 1989. J. Biol. Chem., 264:9258-65; andU.S. Pat. Nos. 5,773,412 and 5,759,996, each of which is incorporatedherein by reference).

It also has been shown in the literature that large peptide cycles canbe substantially shortened, eliminating extraneous amino acids, butsubstantially including the critical binding residues. See, U.S. Pat.No. 5,556,939, incorporated by reference herein.

The shortened cyclic peptides can be formed using disulfide bonds oramide bonds of suitably located carboxylic acid groups and amino groups.

Furthermore, D-amino acids can be added to the peptide sequence tostabilize turn features (especially in the case of glycine). In anotherapproach alpha, beta, gamma or delta dipeptide or turn mimics (such asα, β, γ, or δ turn mimics), some of which are shown in FIGS. 1A-1C, canbe employed to mimic structural motifs and turn features in a peptideand simultaneously provide stability from proteolysis and enhance otherproperties such as, for example, conformational stability and solubility(structure 1A: Hart et al., J. Org. Chem., 64, 2998-2999 (1999);structure 1B: Hanessian et al., “Synthesis of a Versatile PeptidomimeticScaffold” in Methods in Molecular Medicine, Vol. 23: PeptidomimeticsProtocols, W. Kazmierski, Ed. (Humana Press Inc., Totowa, N.J., 1999),Chapter 10, pp. 161-174; structure 1C: WO 01/16135.

Substitution of Disulfide Mimetics

Also within the scope of the invention is the substitution of disulfidemimetics for disulfide bonds within the cMet binding peptides of theinvention.

Where disulfide-containing peptides are employed in generating^(99m)Tc-based radiopharmaceuticals, or other usefulradiopharmaceuticals based on other isotopes, a significant problem isthe presence of the disulfide bond. For example, the integrity of thedisulfide bond is difficult to maintain during procedures designed toincorporate ^(99m)Tc via routes that are reliant upon the reduction ofpertechnetate ion and subsequent incorporation of the reduced Tc speciesinto substances bearing Tc-specific chelating groups. This is becausethe disulfide bond is rather easily reduced by the reducing agentscommonly used in kits devised for one-step preparation ofradiopharmaceuticals. Therefore, the ease with which the disulfide bondcan be reduced during Tc chelation may require substitution withmimetics of the disulfide bonds. Accordingly, another modificationwithin the scope of the invention is to substitute the disulfide moietywith mimetics utilizing the methods disclosed herein or known to thoseskilled in the art, while retaining the activity and other desiredproperties of the cMet-binding polypeptides of the invention.

1.) Oxime Linker

The oxime moiety has been employed as a linker by investigators in anumber of contexts (Wahl, F. and Mutter, M., 1996. Tetrahedron Lett.,37:6861-6864). As shown in FIG. 2, the amino acids 4, containing anaminoalcohol function, and 5 containing an alkoxyamino function, can beincorporated into the peptide chain, not necessarily at the end of thepeptide chain. After formation of the peptide the side-chain protectinggroups can be removed. The aldehyde group is then unmasked and an oximelinkage is formed.

2.) Lanthionine Linker

Lanthionines are cyclic sulfides, wherein the disulfide linkage (S—S) isreplaced by a carbon-sulfur (C—S) linkage. Thus, the lability toreduction is far lower. Lanthionines can be prepared by a number ofmethods including those discussed below.

1) Preparation of Lanthionines using Bromoacetylated Peptides

Lanthionines can be readily prepared using known methods (Robey, F. andFields, R., 1989. Anal. Biochem., 177:373-377; Inman, J. et al., 1991.Bioconjug. Chem., 2:458-463; Ploinsky, A. et al., 1992. J. Med. Chem.,35:4185-4194; Mayer et al., “Peptides, Frontiers of Peptide Science”, inProceedings of the 15th American Peptide Symposium, Tam and Kaumaya(Eds.), Jun. 14-19, 1995, Nashville, Tenn. (Klumer Academic Pub.,Boston), pp. 291-292; Wakao et al., Jpn. Kokai Tokyo Koho, JP 07300452A2 (1995)). Preparation of peptides using Boc automated peptidesynthesis followed by coupling the peptide terminus with bromoaceticacid gives bromoacetylated peptides in good yield. Cleavage anddeprotection of the peptides can be accomplished using HF/anisole. Ifthe peptide contains a cysteine group its reactivity can be controlledwith low pH. If the pH of the medium is raised to 6-7 then eitherpolymerization or cyclization of the peptide takes place. Polymerizationis favored at high (100 mg/mL) concentration whereas cyclization isfavored at lower concentrations (1 mg/mL), e.g., 6 cyclizes to 7(referred to herein as “scheme 1” as shown in FIG. 3). Inman et al.demonstrated the use of Na-(Boc)-Ne-[N-(bromoacetyl)-β-alanyl]R-lysineas a carrier of the bromoacetyl group that could be employed in Bocpeptide synthesis thus allowing placement of a bromoacetyl bearingmoiety anywhere in a sequence. In preliminary experiments they foundthat peptides with 4-6 amino acids separating the bromoacetyl-lysinederivative from a cysteine tend to cyclize, indicating the potentialutility of this strategy.

2) Preparation of Lanthionines Via Cysteine Thiol Addition toAcrylamides

Several variants of this strategy can be implemented. Resin-bound serinecan be employed to prepare the lanthionine ring on resin either using abromination-dehydrobromination-thiol addition sequence or by dehydrationwith disuccinimidyl carbonate followed by thiol addition. Conjugateaddition of thiols to acrylamides has also been amply demonstrated and areference to the addition of 2-mercaptoethanol to acrylamide is provided(Wakao et al., Jpn. Kokai Tokyo Koho, JP 07300452 A2, 1995).

3) Diaryl Ether or Diarylamine Linkage From Intramolecular Cyclizationof Aryl Boronic Acids and Tyrosine

The reaction of arylboronic acids with phenols, amines and heterocyclicamines in the presence of cupric acetate, in air, at ambienttemperature, in dichloromethane using either pyridine or triethylamineas a base to provide unsymmetrical diaryl ethers and the related aminesin good yields (as high as 98%) has been reported (Evans, D. et al.,1998. Tetrahedron Lett., 39:2937-2940; Chan, D. et al., 1998.Tetrahedron Lett., 39:2933-2936; Lam, P. et al., 1998. TetrahedronLett., 39:2941-2944). In the case of N-protected tyrosine derivatives asthe phenol component the yields were also as high as 98%. Thisdemonstrates that amino acid amides (peptides) are expected to be stableto the transformation and that yields are high. Precedent for anintramolecular reaction exists in view of the facile intramolecularcyclizations of peptides to lactams, intramolecular biaryl etherformation based on the SNAr reaction and the generality ofintramolecular cyclization reactions under high dilution conditions oron resin, wherein the pseudo-dilution effect mimics high dilutionconditions.

4) Formation of Cyclic Peptides with a Thiazolidine Linkage viaIntramolecular Reaction of Peptide Aldehydes with Cysteine Moieties

Another approach that may be employed involves intramolecularcyclization of suitably located vicinal amino mercaptan functions(usually derived from placement of a cysteine at a terminus of thelinear sequence or tethered to the sequence via a side-chain nitrogen ofa lysine, for example) and aldehyde functions to provide thiazolidinesthat result in the formation of a bicyclic peptide, one ring of which isthat formed by the residues in the main chain, and the second ring beingthe thiazolidine ring. Scheme 2 (FIG. 4) provides an example. Therequired aldehyde function can be generated by sodium metaperiodatecleavage of a suitably located vicinal aminoalcohol function, which canbe present as an unprotected serine tethered to the chain by appendageto a side chain amino group of a lysine moiety. In some cases therequired aldehyde function is generated by unmasking of a protectedaldehyde derivative at the C-terminus or the N-terminus of the chain(Botti, P. et al., 1996. J. Am. Chem. Soc., 118:10018-10034).

5) Lactams Based on Intramolecular Cyclization of Pendant Amino Groupswith Carboxyl Groups on Resin.

Macrocyclic peptides can be prepared by lactam formation by eitherhead-to-tail or by pendant group cyclization. The basic strategy is toprepare a fully protected peptide wherein it is possible to removeselectively an amine protecting group and a carboxy protecting group.Orthogonal protecting schemes have been developed. Of those that havebeen developed the allyl, trityl and Dde methods have been employed most(Mellor et al., “Synthesis of Modified Peptides”, in Fmoc Solid PhaseSynthesis: A Practical Approach, White and Chan (eds) (Oxford UniversityPress, New York, 2000), Ch. 6, pp. 169-178). The Dde approach is ofinterest because it utilizes similar protecting groups for both thecarboxylic acid function (Dmab ester) and the amino group (Dde group).Both are removed with 2-10% hydrazine in DMF at ambient temperature.Alternately the Dde can be used for the amino group and the allyl groupcan be used for the carboxyl.

A lactam function, available by intramolecular coupling via standardpeptide coupling reagents (such as HATU, PyBOP etc) can act as asurrogate for the disulfide bond. The Dde/Dmab approach is shown inScheme 3 (FIG. 5).

Thus, a linear sequence containing, for example, the Dde-protectedlysine and Dmab ester can be prepared on a Tentagel-based Rink amideresin at low load (˜0.1-0.2 mmol/g). Deprotection of both functions withhydrazine is then followed by on-resin cyclization to give the desiredproducts. Subsequently cleavage from resin and purification may also becarried out. For functionalization of the N-terminus of the peptide itis understood that diamino acids such astrans-4-(iv-Dde)methylaminocyclohexane carboxylic acid or4-(iv-Dde)methylamino benzoic acid would be required. An alternativescenario is to employ the safety catch method to intramolecular lactamformation during cleavage from the resin.

6) Cyclic Peptides Based on Olefin Metathesis

The Grubbs reaction (Scheme 4, FIG. 6) involves themetathesis/cyclization of olefin bonds (Schuster et al., 1997. Angew.Chem. Int. Edn Engl., 36:2036-2056; Miller et al., 1996. J. Am. Chem.Soc., 118:9606-9614). It is readily seen that if the starting materialis a diolefin 16 that the resulting product will be cyclic compound 17.The reaction has been applied to creation of cycles fromolefin-functionalized peptides (Pernerstorfer et al., 1997. Chem.Commun., 20:1949-50; Clark et al., 1999. Chem. Eur. J., 5:782-792;Blackwell et al., 1998 Angew. Chem. Int. Ed., 37:3281-3284; Ripka, A. etal., 1998. Bioorg. Med. Chem. Lett., 8:357-360; Miller et al., 1996. J.Am. Chem. Soc., 118:9606-9614; Clark et al., 1995. J. Am. Chem. Soc.,117:12364-12365; Miller et al., 1995. J. Am. Chem. Soc., 117:5855-5856).One can prepare either C-allylated amino acids or possibly N-allylatedamino acids and employ them in this reaction in order to preparecarba-bridged cyclic peptides as surrogates for disulfide bondcontaining peptides.

One also can prepare novel compounds with olefinic groups.Functionalization of the tyrosine hydroxyl with an olefin-containingtether is one option. The lysine E-amino group is another option withappendage of the olefin-containing unit as part of an acylating moiety,for example. If instead the lysine side chain amino group is alkylatedwith an olefin containing tether, it can still function as a point ofattachment for a reporter as well. The use of 5-pentenoic acid as anacylating agent for the lysine, ornithine, or diaminopropionic sidechain amino groups is another possibility. The length of theolefin-containing tether can also be varied in order to explorestructure activity relationships.

Manipulation of Peptide Sequences

Other modifications within the scope of the invention includemanipulations of peptide sequences, which can be expected to yieldpeptides with similar or improved biological properties. These includeamino acid translocations (swapping amino acids in the sequence), use ofretro-inverso peptides in place of the original sequence or a modifiedoriginal sequence, peptoids and retro-inverso peptoid sequences.Structures wherein specific residues are peptoid instead of peptidic,which result in hybrid molecules, neither completely peptidic norcompletely peptoid, are anticipated as well.

Linkers

Additionally, modifications within the invention include introduction oflinkers or spacers between the targeting sequence of the binding moietyor binding polypeptide and the detectable label or therapeutic agent.For example, use of such linkers/spacers can improve the relevantproperties of the binding peptides (e.g., increase serum stability,etc.). These linkers can include, but are not restricted to, substitutedor unsubstituted alkyl chains, polyethylene glycol derivatives, aminoacid spacers, sugars, or aliphatic or aromatic spacers common in theart.

For example, suitable linkers include homobifunctional andheterobifunctional cross-linking molecules. The homobifunctionalmolecules have at least two reactive functional groups, which are thesame. The reactive functional groups on a homobifunctional moleculeinclude, for example, aldehyde groups and active ester groups.Homobifunctional molecules having aldehyde groups include, for example,glutaraldehyde and subaraldthyde.

Homobifunctional linker molecules having at least two active ester unitsinclude esters of dicarboxylic acids and N-hydroxysuccinimide. Someexamples of such N-succinimidyl esters include disuccinimidyl suberateand dithio-bis-(succinimidyl propionate), and their soluble bis-sulfonicacid and bis-sulfonate salts such as their sodium and potassium salts.

Heterobifunctional linker molecules have at least two different reactivegroups. Some examples of heterobifunctional reagents containing reactivedisulfide bonds include N-succinimidyl 3-(2-pyridyl-dithio)propionate(Carlsson of al., 1978. Biochem. J., 173:723-737), sodiumS-4-succinimidyloxycarbonyl-alpha-methylbenzylthiosulfate, and4-succinimidyloxycarbonyl-alpha-methyl-(2-pyridyldithio)toluene.N-succinimidyl 3-(2-pyridyldithio)propionate is preferred. Some examplesof heterobifunctional reagents comprising reactive groups having adouble bond that reacts with a thiol group include succinimidyl4-(N-maleimidomethyl)cyclohexahe-1-carboxylate and succinimidylm-maleimidobenzoate. Other heterobifunctional molecules includesuccinimidyl 3-(maleimido)propionate, sulfosuccinimidyl4-(p-maleimido-phenyl)butyrate, sulfosuccinimidyl4-(N-maleimidomethyl-cyclohexane)-1-carboxylate,maleimidobenzoyl-5N-hydroxy-succinimide ester.

Furthermore, linkers that are combinations of the molecules and/ormoieties described above, can also be employed to confer specialadvantage to the properties of the peptide. Lipid molecules with linkersmay be attached to allow formulation of ultrasound bubbles, liposomes orother aggregation based constructs. Such constructs could be employed asagents for targeting and delivery of a diagnostic reporter, atherapeutic agent (e.g., a chemical “warhead” for therapy), or acombination of these.

Multimeric Constructs of cMet Binding Polypeptides

Constructs employing dimers, multimers or polymers of one or more cMetbinding polypeptides of the invention are also contemplated. Indeed,there is ample literature evidence that the binding of low potencypeptides or small molecules can be substantially increased by theformation of dimers and multimers. Thus, dimeric and multimericconstructs (both homogeneous and heterogeneous) are within the scope ofthe instant invention. The polypeptide sequences in the dimericconstructs can be attached at their N- or C-terminus or the N-epsilonnitrogen of a suitably placed lysine moiety (or another function bearinga selectively derivatizable group such as a pendant oxyamino or othernucleophilic group), or can be joined together via one or more linkers(e.g., those discussed herein) employing the appropriate attachmentchemistry. This coupling chemistry can include amide, urea, thiourea,oxime, or aminoacetylamide (from chloro- or bromoacetamide derivatives,but is not so limited). For example, methods to prepare dimeric ormultimeric constructs of cMet binding polypeptides of the inventioninclude at least those discussed below.

Method A

Fully protected cMet-binding peptides can be built up on Ellman-typesafety catch resin using automated or manual Fmoc peptide synthesisprotocols (Backes et al., 1996. J. Am. Chem. Soc., 118:3055-56).Separately, using standard methods known in the art of peptidesynthesis, a di-lysine derivative can be constructed on 2-chlorotritylresin (Fields et al., “Principles and Practice of Solid Phase Synthesis”in Synthetic Peptides, A Users Guide, Grant, Ed. (W.H. Freeman Co., NewYork, 1992), Ch. 3, pp. 77-183; Barlos et al., “Convergent PeptideSynthesis” in Fmoc Solid Phase Peptide Synthesis, Chan, W. C. and White,P. D., Eds. (Oxford University Press, New York, 2000), Ch. 9, pp.215-228). Liberation of this from the 2-chlorotrityl resin withoutremoval of the side-chain protecting groups, activation of the carboxylgroup and coupling to any amine-functionalized labeling group provides adi-lysine derivative whose protected pendant nitrogen atoms can beunmasked to give two free amino groups. The prior-mentioned safety-catchresin is activated and the desired N-deprotected labelinggroup-functionalized di-lysine derivative is added to the activatedsafety-catch resin. The pendant amino groups are acylated by thecarboxy-terminus of the safety-catch resin-bound peptide, which is nowdetached from the resin and represents an integral part of the di-lysinestructure. An excess of the safety-catch resin-bound peptide can beemployed to insure complete reaction of the amino groups of thedi-lysine construct. Optimization of the ratio of the reacting partnersin this scheme optimizes the yield. The protecting groups on thecMet-binding peptides are removed employing trifluoroacetic acid basedcleavage protocols.

The synthesis of dimeric and multimeric constructs wherein two or morecMet-binding peptides are present in one construct is easilyaccomplished. Orthogonal protection schemes (such as an allyloxycarbonylgroup on one nitrogen and an Fmoc group on the other, or employing theFmoc group in conjunction with the iV-Dde protecting group on the other,for example) can be employed to distinguish the pendant nitrogen atomsof the di-lysine derivatives described above. Unmasking of one of theamino groups, followed by reaction of the resulting product with anactivated safety-catch resin-bound cMet-binding peptide as describedabove, provides a di-lysine construct having a single cMet-bindingpeptide attached. Removal of the second protecting group unmasks theremaining nitrogen (Mellor et al., “Synthesis of Modified Peptides” inFmoc Solid Phase Peptide Synthesis, Chan, W. C. and White, P. D., Eds.(Oxford University Press, New York, 2000), Chapt. 6, pp. 169-176). Theresulting product can be reacted with a second safety-catch resinbearing another cMet-binding peptide to provide a fully-protectedhomodimeric construct, which after removal of protecting groups withtrifluoroacetic acid, provides the desired material.

Method B

A cMet-binding peptide is assembled on a Rink-amide resin by automatedor manual peptide coupling methods, usually employing Fmoc peptidesynthesis protocols. The peptide can possess a C-terminus or N-terminusfunctionalized with a linker or a linker-labeling group construct thatmay possess an additional nucleophilic group such as the ε-amino groupof a lysine moiety, for example. Cleavage of the protecting groups isaccomplished employing trifluoroacetic acid with appropriate modifiersdepending on the nature of the peptide. The fully deprotected peptide isthen reacted with a large excess of a bifunctional electrophile such asthe commercially available glutaric acid bis-N-hydroxysuccinimide ester(Tyger Scientific, Inc., Princeton, N.J.). The resulting monoamidated,mono-N-hydroxysuccinimidyl ester of glutaric acid is then treated withan additional equivalent of the same peptide, or an equivalent of adifferent cMet-binding peptide. Purification of the resulting materialby HPLC affords the desired homo-dimeric construct bearing a suitablelabeling group.

Method C

A modular scheme can be employed to prepare dimeric or higher multimericconstructs bearing suitable labeling groups as defined above. In asimple illustration, fmoc-lysine(iV-Dde) Rink amide resin is treatedwith piperidine to remove the fmoc moiety. Then a labeling function,such as biotin, 5-carboxyfluorescein orN,N-dimethyl-Gly-Ser(O-t-Bu)-Cys(Acm)-Gly-OH is coupled to the nitrogenatom. The resin is next treated with hydrazine to remove the iV-Ddegroup. After thorough washing, the resin is treated with cyanuricchloride and a hindered base such as diisopropylethylamine in a suitablesolvent such as DMF, NMP or dichloromethane to provide amonofunctionalized dichlorotriazine bound to the resin. Subsequentsuccessive displacement of the remaining chlorine atoms by twoequivalents of a cMet-binding peptide provides a resin-boundhomo-dimeric labeling group-functionalized construct (Falorni, M. etal., 1998. Tetrahedron Lett., 39:7607-7610; Johnson, C. et al., 1998.Tetrahedron, 54:4097-4106; Stankova, M. and Lebl, M., 1996. Mol.Divers., 2:75-80). The incoming peptides can be protected or unprotectedas the situation warrants. Cleavage of protecting groups is accomplishedemploying trifluoroacetic acid-based deprotection reagents as describedabove, and the desired materials are purified by high performance liquidchromatography.

It is understood that in each of these methods lysine derivatives can beserially employed to increase the multiplicity of the multimers. The useof related, more rigid molecules bearing the requisite number of masked,or orthogonally protected nitrogen atoms to act as scaffolds to vary thedistance between the cMet-binding peptides, to increase the rigidity ofthe construct (by constraining the motion and relative positions of thecMet-binding peptides relative to each other and the reporter) isentirely within the scope of methods A-C and all other methods describedherein. The references cited above are incorporated by reference hereinin their entirety.

Uses for cMet Binding Polypeptides and Multimeric Peptide Constructs

The cMet binding moieties of the invention also have utility in thetreatment of a variety of disease states, including those associatedwith cellular proliferation (e.g., hyperproliferation, e.g., cancer).The cMet binding moieties of the invention (e.g., polypeptides andmultimeric polypeptide constructs) can themselves be used astherapeutics or could be used to localize one or more therapeutic agents(e.g., a chemotherapeutic, a radiotherapeutic, genetic material, etc.)to cMet-expressing cells, including sites of cellular proliferation. Anysuitable method of assaying or imaging cMet also can be employed. ThecMet binding moieties according to this invention are useful fordetection and/or imaging of cMet in vitro or in vivo, and particularlyfor detection and/or imaging of sites of angiogenesis, in which HGF andcMet are intimately involved, as explained herein.

In Vitro

For detection of HGF or cMet in solution, a binding polypeptide ormultimeric polypeptide construct according to the invention can bedetectably labeled, e.g., fluorescently labeled, enzymatically labeled,or labeled with a radioactive or paramagnetic metal, then contacted withthe solution, and thereafter formation of a complex between the bindingpolypeptide and the cMet target can be detected. As an example, afluorescently labeled cMet binding peptide can be used for in vitro cMetor HGF/cMet complex detection assays, wherein the peptide is added to asolution to be tested for cMet or HGF/cMet complex under conditionsallowing binding to occur. The complex between the fluorescently labeledcMet binding peptide and cMet or HGF/cMet complex target can be detectedand quantified by, for example, measuring the increased fluorescencepolarization arising from the cMet or HGF/cMet complex-bound peptiderelative to that of the free peptide.

Alternatively, a sandwich-type “ELISA” assay can be used, wherein a cMetbinding polypeptide is immobilized on a solid support such as a plastictube or well, then the solution suspected of containing cMet or HGF/cMetcomplex target is contacted with the immobilized binding moiety,non-binding materials are washed away, and complexed polypeptide isdetected using a suitable detection reagent, such as a monoclonalantibody recognizing cMet or HGF/cMet complex. The monoclonal antibodyis detectable by conventional means known in the art, including beingdetectably labeled, e.g., radiolabeled, conjugated with an enzyme suchas horseradish peroxidase and the like, or fluorescently labeled, etc.

For detection or purification of soluble cMet or HGF/cMet complex in orfrom a solution, binding polypeptides or multimeric polypeptideconstruct of the invention can be immobilized on a solid substrate suchas a chromatographic support or other matrix material, then theimmobilized binder can be loaded or contacted with the solution underconditions suitable for formation of a binding polypeptide/cMet complex.The non-binding portion of the solution can be removed and the complexcan be detected, for example, using an anti-HGF or anti-HGF/cMet complexantibody, or an anti-binding polypeptide antibody, or the cMet orHGF/cMet complex target can be released from the binding moiety atappropriate elution conditions.

The biology of cellular proliferation and the roles of HGF and cMet ininitiating and maintaining it have been investigated by many researchersand continues to be an active field for research and development. Infurtherance of such research and development, a method of purifying bulkamounts of cMet or HGF/cMet complex in pure form is desirable, and thebinding polypeptides and multimeric polypeptide constructs according tothis invention are especially useful for that purpose, using the generalpurification methodology described above.

In Vivo Diagnostic Imaging

A particularly preferred use for the polypeptides and multimericpolypeptide constructs according to the present invention is forcreating visually readable images of cMet expressing tissue, such as,for example, neoplastic tumors, which exhibit hyperproliferation. ThecMet binding polypeptides and multimeric polypeptide constructsdisclosed herein can be converted to imaging reagents by conjugating thepolypeptides with a label appropriate for diagnostic detection,optionally via a linker. Preferably, a peptide or multimeric polypeptideconstruct exhibiting much greater specificity for cMet or HGF/cMet thanfor other serum proteins is conjugated or linked to a label appropriatefor the detection methodology to be employed. For example, the cMet orHGF/cMet complex binding polypeptide can be conjugated with or without alinker to a paramagnetic chelate suitable for Magnetic Resonance Imaging(MRI), with a radiolabel suitable for x-ray, Positron EmissionTomography (PET) or scintigraphic imaging (including a chelator for aradioactive metal), with an ultrasound contrast agent (e.g., astabilized microbubble, a microballoon, a microsphere or what has beenreferred to as a gas filled “liposome”) suitable for ultrasounddetection, or with an optical imaging dye.

Suitable linkers can include those discussed herein, includingsubstituted or unsubstituted alkyl chains, amino acid chains (e.g.,polyglycine), polyethylene glycols, polyamides, and other linkers knownin the art.

In general, the technique of using a detectably labeled cMet bindingmoiety is based on the premise that the label generates a signal that isdetectable outside a patient's body. For example, when the detectablylabeled cMet binding moiety is administered to the patient in which itis desirable to detect, e.g., hyperproliferation, the high affinity ofthe cMet binding moiety for cMet causes the binding moiety to bind tothe site of hyperproliferation and accumulate label at the site.Sufficient time is allowed for the labeled binding moiety to localize atthe site of proliferation. The signal generated by the labeled peptideis detected by a scanning device that will vary according to the type oflabel used, and the signal is then converted to an image of the site ofproliferation.

In another embodiment, rather than directly labeling a cMet bindingpolypeptide or multimeric polypeptide construct with a detectable labelor radiotherapeutic construct, one or more peptides or constructs of theinvention can be conjugated with for example, avidin, biotin, or anantibody or antibody fragment that will bind the detectable label orradiotherapeutic. For example, one or more cMet-binding peptides can beconjugated to streptavidin (potentially generating multivalent binding)for in vivo binding to cMet-expressing cells. After the unboundtargeting construct is cleared from the body, a biotinylated detectablelabel or radiotherapeutic construct (e.g., a chelate molecule complexedwith a radioactive metal) can be infused and will rapidly concentrate atthe site where the targeting construct is bound. This approach in somesituations can reduce the time required after administering thedetectable label until imaging can take place. It also can increasesignal to noise ratio in the target site, and decrease the dose of thedetectable label or radiotherapeutic construct required. This isparticularly useful when a radioactive label or radiotherapeutic is usedas the dose of radiation that is delivered to normal butradiation-sensitive sites in the body, such as bone-marrow, kidneys, andliver is decreased. This approach, sometimes referred to aspre-targeting or two-step, or three-step approaches was reviewed by S.F. Rosebrough in Q. J. Nucl. Med., 40:234-251 (1996), which isincorporated by reference herein.

A. Magnetic Resonance Imaging

The cMet binding moieties of the present invention can advantageously beconjugated with a paramagnetic metal chelate in order to form a contrastagent for use in MRI. Preferred paramagnetic metal ions have atomicnumbers 21-29, 42, 44, or 57-83. This includes ions of the transitionmetal or lanthanide series which have one, and more preferably five ormore, unpaired electrons and a magnetic moment of at least 1.7 Bohrmagneton. Preferred paramagnetic metals include, but are not limited to,chromium (III), manganese (II), manganese (III), iron (II), iron (III),cobalt (II), nickel (II), copper (II), praseodymium (III), neodymium(III), samarium (III), gadolinium (III), terbium (III), dysprosium(III), holmium erbium (III), europium (III) and ytterbium (III),chromium (III), iron (III), and gadolinium (III). The trivalent cation,Gd³⁺, is particularly preferred for MRI contrast agents, due to its highrelaxivity and low toxicity, with the further advantage that it existsin only one biologically accessible oxidation state, which minimizesundesired metabolysis of the metal by a patient. Another useful metal isCr³⁺, which is relatively inexpensive. Gd(III) chelates have been usedfor clinical and radiologic MR applications since 1988, andapproximately 30% of MR exams currently employ a gadolinium-basedcontrast agent.

The practitioner will select a metal according to dose required todetect cellular proliferation and considering other factors such astoxicity of the metal to the subject. See, Tweedle et al., MagneticResonance Imaging (2nd ed.), vol. 1, Partain et al., Eds. (W.B. SaundersCo. 1988), pp. 796-797. Generally, the desired dose for an individualmetal will be proportional to its relaxivity, modified by thebiodistribution, pharmacokinetics and metabolism of the metal.

The paramagnetic metal chelator is a molecule having one or more polargroups that act as a ligand for, and complex with, a paramagnetic metal.Suitable chelators are known in the art and include acids with methylenephosphonic acid groups, methylene carbohydroxarnine acid groups,carboxyethylidene groups, or carboxymethylene groups. Examples ofchelators include, but are not limited to, diethylenetriaminepentaaceticacid (DTPA), 1,4,7,10-tetraazacyclo-tetradecane-1,4,7,10-tetraaceticacid (DOTA), 1-substituted1,4,7,-tricarboxymethyl-1,4,7,10-teraazacyclododecane (DO3A),ethylenediaminetetraacetic acid (EDTA), and1,4,8,11-tetra-azacyclotetradecane-1,4,8,11-tetraacetic acid (TETA).Additional chelating ligands are ethylene bis-(2-hydroxy-phenylglycine)(EHPG), and derivatives thereof, including 5-C1-EHPG, 5-Br-EHPG,5-Me-EHPG, 5-t-Bu-EHPG, and 5-sec-Bu-EHPG; benzodiethylenetriaminepentaacetic acid (benzo-DTPA) and derivatives thereof, includingdibenzo-DTPA, phenyl-DTPA, diphenyl-DTPA, benzyl-DTPA, and dibenzylDTPA; bis-2 (hydroxybenzyl)-ethylene-diaminediacetic acid (HBED) andderivatives thereof; the class of macrocyclic compounds which contain atleast 3 carbon atoms, more preferably at least 6, and at least twoheteroatoms (0 and/or N), which macrocyclic compounds can consist of onering, or two or three rings joined together at the hetero ring elements,e.g., benzo-DOTA, dibenzo-DOTA, and benzo-NOTA, where NOTA is1,4,7-triazacyclononane N,N′,N″-triacetic acid, benzo-TETA, benzo-DOTMA,where DOTMA is 1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetra(methyltetraacetic acid), and benzo-TETMA, where TETMA is1,4,8,11-tetraazacyclotetradecane-1,4,8,11-(methyl tetraacetic acid);derivatives of 1,3-propylene-diaminetetraacetic acid (PDTA) andtriethylenetetraaminehexaacetic acid (TTNA); derivatives of1,5,10?N,N,N″-tris(2,3-dihydroxybenzoyl)-tricatecholate (LICAM); and1,3,5-N,N′,N″-tris(2,3-dihydroxybenzoyl)aminomethylbenzene (MECAM). Apreferred chelator for use in the present invention is DTPA, and the useof DO3A is particularly preferred. Examples of representative chelatorsand chelating groups contemplated by the present invention are describedin WO 98/18496, WO 86/06605, WO 91/03200, WO 95/28179, WO 96/23526, WO97/36619, PCT/US98/01473, PCT/US98/20182, and U.S. Pat. No. 4,899,755,U.S. Pat. No. 5,474,756, U.S. Pat. No. 5,846,519 and U.S. Pat. No.6,143,274, all of which are hereby incorporated by reference.

In accordance with the present invention, the chelator of the MRIcontrast agent is coupled to the cMet binding polypeptide. Thepositioning of the chelate should be selected so as not to interferewith the binding affinity or specificity of the cMet bindingpolypeptide. Preferably, the chelate will be appended either to theN-terminus or the C-terminus, however the chelate also can be attachedanywhere within the sequence. In preferred embodiments, a chelatorhaving a free central carboxylic acid group (e.g.,DTPA-Asp(β-COOH)-)OtBu) makes it easy to attach at the N-terminus of thepeptide by formation of an amide bond. The chelate also can be attachedat the C-terminus with the aid of a linker. Alternatively,isothiocyanate conjugation chemistry can be employed as a way of linkingthe appropriate isothiocyanate group bearing DTPA to a free amino groupanywhere within the peptide sequence.

In general, the cMet binding moiety can be bound directly or covalentlyto the metal chelator (or other detectable label), or it can be coupledor conjugated to the metal chelator using a linker, which can be,without limitation, amide, urea, acetal, ketal, double ester, carbonyl,carbamate, thiourea, sulfone, thioester, ester, ether, disulfide,lactone, imine, phosphoryl, or phosphodiester linkages; substituted orunsubstituted saturated or unsaturated alkyl chains; linear, branched,or cyclic amino acid chains of a single amino acid or different aminoacids (e.g., extensions of the N- or C-terminus of the cMet bindingmoiety); derivatized or underivatized polyethylene glycols (PEGS),polyoxyethylene, or polyvinylpyridine chains; substituted orunsubstituted polyamide chains; derivatized or underivatized polyamine,polyester, polyethylenimine, polyacrylate, poly(vinyl alcohol),polyglycerol, or oligosaccharide (e.g., dextran) chains; alternatingblock copolymers; malonic, succinic, glutaric, adipic and pimelic acids;caproic acid; simple diamines and dialcohols; any of the other linkersdisclosed herein; or any other simple polymeric linkers known in the art(see, for example, WO 98/18497 and WO 98/18496). Preferably themolecular weight of the linker can be tightly controlled. The molecularweights can range in size from less than 100 to greater than 1000.Preferably the molecular weight of the linker is less than 100. Inaddition, it can be desirable to utilize a linker that is biodegradablein vivo to provide efficient routes of excretion for the imagingreagents of the present invention. Depending on their location withinthe linker, such biodegradable functionalities can include ester, doubleester, amide, phosphoester, ether, acetal, and ketal functionalities.

In general, known methods can be used to couple the metal chelate andthe cMet binding moiety using such linkers (WO 95/28967, WO 98/18496, WO98/18497 and discussion therein). The cMet binding moiety can be linkedthrough an N- or C-terminus via an amide bond, for example, to a metalcoordinating backbone nitrogen of a metal chelate or to an acetate armof the metal chelate itself. The present invention contemplates linkingof the chelate on any position, provided the metal chelate retains theability to bind the metal tightly in order to minimize toxicity.Similarly, the cMet binding moiety can be modified or elongated in orderto generate a locus for attachment to a metal chelate, provided suchmodification or elongation does not eliminate its ability to bind cMet.

MRI contrast reagents prepared according to the disclosures herein canbe used in the same manner as conventional MRI contrast reagents. Whenimaging a site of hyperproliferation, for example, certain MR techniquesand pulse sequences can be preferred to enhance the contrast of the siteto the background blood and tissues. These techniques include (but arenot limited to), for example, black blood angiography sequences thatseek to make blood dark, such as fast spin echo sequences (Alexander, A.et al., 1998. Magn. Reson. Med., 40: 298-310) and flow-spoiled gradientecho sequences (Edelman, R. et al., 1990. Radiology, 177: 45-50). Thesemethods also include flow independent techniques that enhance thedifference in contrast, such as inversion-recovery prepared orsaturation-recovery prepared sequences that will increase the contrastbetween angiogenic tumor and background tissues. Finally, magnetizationtransfer preparations also can improve contrast with these agents(Goodrich, K. et al., 1996. Invest. Radial., 31: 323-32).

The labeled reagent is administered to the patient in the form of aninjectable composition. The method of administering the MRI contrastagent is preferably parenterally, meaning intravenously,intraarterially, intrathecally, interstitially, or intracavitarilly. Forimaging active angiogenesis, intravenous or intraarterial administrationis preferred. For MRI, it is contemplated that the subject will receivea dosage of contrast agent sufficient to enhance the MR signal at thesite of angiogenesis at least 10%. After injection with the cMet bindingmoiety-containing MRI reagent, the patient is scanned in the MRI machineto determine the location of any sites of hyperproliferation. Intherapeutic settings, upon identification of a site ofhyperproliferation (e.g., tumor), a tumoricidal agent oranti-hyperproliferative agent (e.g., inhibitors of HGF) can beimmediately administered, if necessary, and the patient can besubsequently scanned to visualize tumor regression or arrest ofangiogenesis.

B. Ultrasound Imaging

When ultrasound is transmitted through a substance, the acousticproperties of the substance will depend upon the velocity of thetransmissions and the density of the substance. Changes in the acousticproperties will be most prominent at the interface of differentsubstances (solids, liquids, gases). Ultrasound contrast agents areintense sound wave reflectors because of the acoustic differencesbetween the agent and the surrounding tissue. Gas containing or gasgenerating ultrasound contrast agents are particularly useful because ofthe acoustic difference between liquid (e.g., blood) and thegas-containing or gas generating ultrasound contrast agent. Because oftheir size, ultrasound contrast agents comprising microbubbles,microballoons, and the like can remain for a longer time in the bloodstream after injection than other detectable moieties; a targetedcMet-specific ultrasound agent therefore could demonstrate superiorimaging of sites of hyperproliferation (e.g., cancer) and angiogenesis.

In this aspect of the invention, the cMet binding moiety can be linkedto a material that is useful for ultrasound imaging. For example, one ormore cMet binding polypeptide or multimeric polypeptide constructs canbe linked to materials employed to form vesicles (e.g., microbubbles,microballoons, microspheres, etc.), or emulsions containing a liquid orgas, which functions as the detectable label (e.g., an echogenic gas ormaterial capable of generating an echogenic gas). Materials for thepreparation of such vesicles include surfactants, lipids, sphingolipids,oligolipids, phospholipids, proteins, polypeptides, carbohydrates, andsynthetic or natural polymeric materials (WO 98/53857, WO 98/18498, WO98/18495, WO 98/18497, WO 98/18496, and WO 98/18501, incorporated hereinby reference in their entirety).

For contrast agents comprising suspensions of stabilized microbubbles (apreferred embodiment), phospholipids, and particularly saturatedphospholipids are preferred. Examples of suitable phospholipids includeesters of glycerol with one or two (the same or different) fatty acidsmolecules and with phosphoric acid, wherein the phosphoric acid residueis in turn bonded to a hydrophilic group, such as choline, serine,inositol, glycerol, ethanolamine, and the like groups. Fatty acidspresent in the phospholipids are in general long chain aliphatic acids,typically containing from 12 to 24 carbon atoms, preferably from 14 to22, that can be saturated or can contain one or more unsaturations.Examples of suitable fatty acids are lauric acid, myristic acid,palmitic acid, stearic acid, arachidonic acid, behenic acid, oleic acid,linoleic acid, and linolenic acid. Mono esters of phospholipid are alsoknown in the art as the “lyso” forms of the phospholipids. Furtherexamples of phospholipid are phosphatidic acids, i.e., the diesters ofglycerol-phosphoric acid with fatty acids, sphingomyelins, i.e., thosephosphatidylcholine analogs where the residue of glycerol diester withfatty acids is replaced by a ceramide chain, cardiolipins, i.e., theesters of 1,3-diphosphatidylglycerol with a fatty acid, gangliosides,cerebrosides, etc.

As used herein, the term “phospholipids” includes naturally occurring,semisynthetic or synthetically prepared products that can be employedeither singularly or as mixtures.

Examples of naturally occurring phospholipids are natural lecithins(phosphatidylcholine (PC) derivatives) such as, typically, soya bean oregg yolk lecithins. Examples of semisynthetic phospholipids are thepartially or fully hydrogenated derivatives of the naturally occurringlecithins.

Examples of synthetic phospholipids are, e.g.,dilauryloyl-phosphatidylcholine (“DLPC”), dimyristoylphosphatidylcholine(“DMPC”), dipalmitoyl-phosphatidylcholine (“DPPC”),diarachidoylphosphatidylcholine (“DAPC”), distearoyl-phosphatidylcholine(“DSPC”), 1-myristoyl-2-palmitoylphosphatidylcholine (“MPPC”),1-pahnitoyl-2-myristoylphosphatidylcholine (“PMPC”),1-palmitoyl-2-stearoylphosphatid-ylcholine (“PSPC”),1-stearoyl-2-palmitoyl-phosphatidylcholine (“SPPC”),dioleoylphosphatidylycholine (“DOPC”), 1,2Distearoyl-sn-glycero-3-Ethylphosphocholine (Ethyl-DSPC),dilauryloyl-phosphatidylglycerol (“DLPG”) and its alkali metal salts,diarachidoylphosphatidylglycerol (“DAPG”) and its alkali metal salts,dimyristoylphosphatidylglycerol (“DMPG”) and its alkali metal salts,dipalmitoyl phosphatidylglycerol (“DPPG”) and its alkali metal salts,distearolyphosphatidylglycerol (“DSPG”) and its alkali metal salts,dioleoylphosphatidylglycerol (“DOPG”) and its alkali metal salts,dimyristoyl phosphatidic acid (“DMPA”) and its alkali metal salts,dipalmitoyl phosphatidic acid (“DPPA”) and its alkali metal salts,distearoyl phosphatidic acid (“DSPA”), diarachidoyl phosphatidic acid(“DAPA”) and its alkali metal salts, dimyristoylphosphatidyl-ethanolamine (“DMPE”), dipalmitoyl phosphatidylethanolamine(“DPPE”), distearoyl phosphatidyl-ethanolamine (“DSPE”), dimyristoylphosphatidylserine (“DMPS”), diarachidoyl phosphatidylserine (“DAPS”),dipalmitoyl phosphatidylserine (“DPPS”), distearoylphosphatidylserine(“DSPS”), dioleoylphosphatidylserine (“DOPS”), dipalmitoyl sphingomyelin(“DPSP”), and distearoyl sphingomyelin (“DSSP”). In a preferredembodiment, at least one of the phospholipid moieties has the structureshown in FIG. 7A or 7B, and described in U.S. Pat. No. 5,686,060, whichis herein incorporated by reference.

Other preferred phospholipids include dipalmitoylphosphatidylcholine,dipalmitoylphosphatidic acid and dipalmitoylphosphatidylserine. Thecompositions also can contain PEG-4000 and/or palmitic acid. Any of thegases disclosed herein or known to the skilled artisan can be employed;however, inert gases, such as SF6 or fluorocarbons like CF4, C3F8 andC4F10, are preferred.

The preferred gas-filled microbubbles of the invention can be preparedby means known in the art, such as, for example, by a method describedin any one of the following patents: EP 554213, U.S. Pat. No. 5,413,774,U.S. Pat. No. 5,578,292, EP 744962, EP 682530, U.S. Pat. No. 5,556,610,U.S. Pat. No. 5,846,518, U.S. Pat. No. 6,183,725, EP 474833, U.S. Pat.No. 5,271,928, U.S. Pat. No. 5,380,519, U.S. Pat. No. 5,531,980, U.S.Pat. No. 5,567,414, U.S. Pat. No. 5,658,551, U.S. Pat. No. 5,643,553,U.S. Pat. No. 5,911,972, U.S. Pat. No. 6,110,443, U.S. Pat. No.6,136,293, EP 619743, U.S. Pat. No. 5,445,813, U.S. Pat. No. 5,597,549,U.S. Pat. No. 5,686,060, U.S. Pat. No. 6,187,288, and U.S. Pat. No.5,908,610, which are incorporated by reference herein in their entirety.

The preferred microbubble suspensions of the present invention can beprepared from phospholipids using known processes such as afreeze-drying or spray-drying solutions of the crude phospholipids in asuitable solvent or using the processes set forth in EP 554213; U.S.Pat. No. 5,413,774; U.S. Pat. No. 5,578,292; EP 744962; EP 682530; U.S.Pat. No. 5,556,610; U.S. Pat. No. 5,846,518; U.S. Pat. No. 6,183,725; EP474833; U.S. Pat. No. 5,271,928; U.S. Pat. No. 5,380,519; U.S. Pat. No.5,531,980; U.S. Pat. No. 5,567,414; U.S. Pat. No. 5,658,551; U.S. Pat.No. 5,643,553; U.S. Pat. No. 5,911,972; U.S. Pat. No. 6,110,443; U.S.Pat. No. 6,136,293; EP 619743; U.S. Pat. No. 5,445,813; U.S. Pat. No.5,597,549; U.S. Pat. No. 5,686,060; U.S. Pat. No. 6,187,288; and U.S.Pat. No. 5,908,610, which are incorporated by reference herein in theirentirety. Most preferably, the phospholipids are dissolved in an organicsolvent and the solution is dried without going through a liposomeformation stage. This can be done by dissolving the phospholipids in asuitable organic solvent together with a hydrophilic stabilizersubstance or a compound soluble both in the organic solvent and waterand freeze-drying or spray-drying the solution. In this embodiment thecriteria used for selection of the hydrophilic stabilizer is itssolubility in the organic solvent of choice. Examples of hydrophilicstabilizer compounds soluble in water and the organic solvent are, e.g.,a polymer, like polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA),polyethylene glycol (PEG), etc., malic acid, glycolic acid, maltol, andthe like. Such hydrophilic compounds also aid in homogenizing themicrobubbles size distribution and enhance stability under storage. Anysuitable organic solvent can be used as long as its boiling point issufficiently low and its melting point is sufficiently high tofacilitate subsequent drying. Typical organic solvents include, forexample, dioxane, cyclohexanol, tertiary butanol, tetrachlorodifluoroethylene (C₂Cl₄F₂) or 2-methyl-2-butanol. 2-methyl-2-butanol and C₂Cl₄F₂are preferred.

Prior to formation of the suspension of microbubbles by dispersion in anaqueous carrier, the freeze dried or spray dried phospholipid powdersare contacted with air or another gas. When contacted with the aqueouscarrier the powdered phospholipids whose structure has been disruptedwill form lamellarized or laminarized segments that will stabilize themicrobubbles of the gas dispersed therein. This method permitsproduction of suspensions of microbubbles, which are stable even whenstored for prolonged periods, and are obtained by simple dissolution ofthe dried laminarized phospholipids, which have been stored under adesired gas, without shaking or any violent agitation.

Unless it contains a hyperpolarized gas, known to require specialstorage conditions, the lyophilized or freeze-dried residue can bestored and transported without need of temperature control of itsenvironment and in particular it can be supplied to hospitals andphysicians for on site formulation into a ready-to-use administrablesuspension without requiring such users to have special storagefacilities.

Preferably in such a case it can be supplied in the form of a twocomponent kit. The two component kit can include two separate containersor a dual-chamber container. In the former case preferably the containeris a conventional septum-sealed vial, wherein the vial containing thelyophilized residue of step b) is sealed with a septum through which thecarrier liquid can be injected using an optionally pre-filled syringe.In such a case the syringe used as the container of the second componentis also used then for injecting the contrast agent. In the latter case,preferably the dual-chamber container is a dual-chamber syringe and oncethe lyophilizate/freeze-dried residue has been reconstituted and thensuitably mixed or gently shaken, the container can be used directly forinjecting the contrast agent. In both cases means for directing orpermitting application of sufficient bubble forming energy into thecontents of the container are provided. However, as noted above, in thestabilized contrast agents the size of the gas microbubbles issubstantially independent of the amount of agitation energy applied tothe reconstituted dried product. Accordingly no more than gentle handshaking is generally required to give reproducible products withconsistent microbubble size.

It can be appreciated by one ordinary skilled in the art that othertwo-chamber reconstitution systems capable of combining the dried powderwith the aqueous solution in a sterile manner are also within the scopeof the present invention. In such systems, it is particularlyadvantageous if the aqueous phase can be interposed between thewater-insoluble gas and the environment, to increase shelf life of theproduct. Where a material necessary for forming the contrast agent isnot already present in the container (e.g., a cMet binding moiety of theinvention to be linked to the phospholipid during reconstitution), itcan be packaged with the other components of the kit, preferably in aform or container adapted to facilitate ready combination with the othercomponents of the kit.

No specific containers, vial or connection systems are required; thepresent invention can use conventional containers, vials and adapters.The only requirement is a good seal between the stopper and thecontainer. The quality of the seal, therefore, becomes a matter ofprimary concern; any degradation of seal integrity could allowundesirables substances to enter the vial. In addition to assuringsterility, vacuum retention is essential for products stoppered atambient or reduced pressures to assure safe and proper reconstitution.As to the stopper, it may be a compound or multicomponent formulationbased on an elastomer, such as poly(isobutylene) or butyl rubber.

Alternatively, microbubbles can be prepared by suspending a gas in anaqueous solution at high agitation speed, as disclosed, e.g., in WO97/29783. A further process for preparing microbubbles is disclosed inco-pending European patent application no. 03002373, herein incorporatedby reference, which comprises preparing an emulsion of an organicsolvent in an aqueous medium in the presence of a phospholipid andsubsequently lyophilizing said emulsion, after optional washing and/orfiltration steps.

Additives known to those of ordinary skill in the art can be included inthe suspensions of stabilized microbubbles. For instance, non-filmforming surfactants, including polyoxypropylene glycol andpolyoxyethylene glycol and similar compounds, as well as variouscopolymers thereof; fatty acids such as myristic acid, palmitic acid,stearic acid, arachidonic acid or their derivatives, ergosterol,phytosterol, sitosterol, lanosterol, tocopherol, propyl gallate,ascorbyl palmitate and butylated hydroxytoluene may be added. The amountof these non-film forming surfactants is usually up to 50% by weight ofthe total amount of surfactants but preferably between 0 and 30%.

In ultrasound applications the contrast agents formed by phospholipidstabilized microbubbles can, for example, be administered in doses suchthat the amount of phospholipid injected is in the range 0.1 to 200μg/kg body weight, preferably from about 0.1 to 30 μg/kg.

Other gas containing suspensions include those disclosed in, forexample, U.S. Pat. No. 5,798,091, WO 97/29783, also EP 881 915,incorporated herein by reference in their entirety. These agents can beprepared as described in U.S. Pat. No. 5,798,091 or WO97/29783.

Another preferred ultrasound contrast agent comprises microballoons. Theterm “microballoon” refers to gas filled bodies with a material boundaryor envelope. More on microballoon formulations and methods ofpreparation can be found in EP 324 938 (U.S. Pat. No. 4,844,882); U.S.Pat. No. 5,711,933; U.S. Pat. No. 5,840,275; U.S. Pat. No. 5,863,520;U.S. Pat. No. 6,123,922; U.S. Pat. No. 6,200,548; U.S. Pat. No.4,900,540; U.S. Pat. No. 5,123,414; U.S. Pat. No. 5,230,882; U.S. Pat.No. 5,469,854; U.S. Pat. No. 5,585,112; U.S. Pat. No. 4,718,433; U.S.Pat. No. 4,774,958; WO 95/01187; U.S. Pat. No. 5,529,766; U.S. Pat. No.5,536,490; and U.S. Pat. No. 5,990,263, the contents of which areincorporated herein by reference.

The preferred microballoons have an envelope including a biodegradablephysiologically compatible polymer or, a biodegradable solid lipid. Thepolymers useful for the preparation of the microballoons of the presentinvention can be selected from the biodegradable physiologicallycompatible polymers, such as any of those described in any of thefollowing patents: EP 458745; U.S. Pat. No. 5,711,933; U.S. Pat. No.5,840,275; EP 554213; U.S. Pat. No. 5,413,774; and U.S. Pat. No.5,578,292, the entire contents of which are incorporated herein byreference. In particular, the polymer can be selected from biodegradablephysiologically compatible polymers, such as polysaccharides of lowwater solubility, polylactides and polyglycolides and their copolymers,copolymers of lactides and lactones such as ε-caprolactone,γ-valerolactone and polypeptides. Other suitable polymers includepoly(ortho)esters (see for instance U.S. Pat. No. 4,093,709; U.S. Pat.No. 4,131,648; U.S. Pat. No. 4,138,344; U.S. Pat. No. 4,180,646);polylactic and polyglycolic acid and their copolymers, for instanceDEXON (Heller, J., 1980. Biomaterials, 1:51-57);poly(DL-lactide-co-e-caprolactone), poly(DL-lactide-co-γ-valerolactone),poly(DL-lactide-co-γ-butyrolactone), polyalkylcyanoacrylates;polyamides, polyhydroxybutyrate; polydioxanone; poly-β-aminoketones(Polymer, 23:1693 (1982)); polyphosphazenes (Allcock, H., 1976. Science,193:1214-1219); and polyanhydrides. The microballoons of the presentinvention can also be prepared according to the methods of WO 96/15815,incorporated herein by reference, where the microballoons are made froma biodegradable membrane comprising biodegradable lipids, preferablyselected from mono- di-, tri-glycerides, fatty acids, sterols, waxes andmixtures thereof. Preferred lipids are di- or tri-glycerides, e.g. di-or tri-myristin, -palmityn or -stearin, in particular tripalmitin ortristearin.

The microballoons can employ any of the gases disclosed herein of knownto the skilled artisan; however, inert gases such as fluorinated gasesare preferred. The microballoons can be suspended in a pharmaceuticallyacceptable liquid carrier with optional additives known to those ofordinary skill in the art and stabilizers.

Microballoons-containing contrast agents are typically administered indoses such that the amount of wall-forming polymer or lipid is fromabout 10 μg/kg to about 20 μg/kg of body weight.

Other gas-containing contrast agent formulations include microparticles(especially aggregates of microparticles) having gas contained thereinor otherwise associated therewith (for example being adsorbed on thesurface thereof and/or contained within voids, cavities or porestherein). Methods for the preparation of these agents are as describedin EP 0122624; EP 0123235; EP 0365467; U.S. Pat. No. 5,558,857; U.S.Pat. No. 5,607,661; U.S. Pat. No. 5,637,289; U.S. Pat. No. 5,558,856;U.S. Pat. No. 5,137,928; WO 95/21631 or WO 93/13809, incorporated hereinby reference in their entirety.

Any of these ultrasound compositions also should be, as far as possible,isotonic with blood. Hence, before injection, small amounts of isotonicagents can be added to any of above ultrasound contrast agentsuspensions. The isotonic agents are physiological solutions commonlyused in medicine and they comprise aqueous saline solution (0.9% NaCl),2.6% glycerol solution, 5% dextrose solution, etc. Additionally, theultrasound compositions can include standard pharmaceutically acceptableadditives, including, for example, emulsifying agents, viscositymodifiers, cryoprotectants, lyoprotectants, bulking agents etc.

Any biocompatible gas can be used in the ultrasound contrast agentsuseful in the invention. The term “gas” as used herein includes anysubstances (including mixtures) substantially in gaseous form at thenormal human body temperature. The gas may thus include, for example,air, nitrogen, oxygen, CO₂, argon, xenon or krypton, fluorinated gases(including for example, perfluorocarbons, SF₆, SeF₆) a low molecularweight hydrocarbon (e.g., containing from 1 to 7 carbon atoms), forexample, an alkane such as methane, ethane, a propane, a butane or apentane, a cycloalkane such as cyclopropane, cyclobutane orcyclopentene, an alkene such as ethylene, propene, propadiene or abutene, or an alkyne such as acetylene or propyne and/or mixturesthereof. However, fluorinated gases are preferred. Fluorinated gasesinclude materials which contain at least one fluorine atom such as SF₆,freons (organic compounds containing one or more carbon atoms andfluorine, i.e., CF₄, C₂F₆, C₃F₈, C₄F₈, C₄F₁₀, CBrF₃, CCl₂F₂, C₂ClF₅, andCBrClF₂) and perfluorocarbons. The term perfluorocarbon refers tocompounds containing only carbon and fluorine atoms and includes, inparticular, saturated, unsaturated, and cyclic perfluorocarbons. Thesaturated perfluorocarbons, which are usually preferred, have theformula CF_(n+2), where n is from 1 to 12, preferably from 2 to 10, mostpreferably from 3 to 8 and even more preferably from 3 to 6. Suitableperfluorocarbons include, for example, CF₄, C₂F₆, C₃F₈ C₄F₈, C₄F₁₀,C₅F₁₂, C₆F₁₂, C₇F₁₄, C₈F₁₈, and C₉F₂₀. Most preferably the gas or gasmixture comprises SF6 or a perfluorocarbon selected from the groupconsisting of C₃F₈ C₄F₈, C₄F₁₀, C₆F₁₂, C₆F₁₂, C₇F₁₄, C₈F₁₈, with C₄F₁₀being particularly preferred. See also WO 97/29783, WO 98/53857, WO98/18498, WO 98/18495, WO 98/18496, WO 98/18497, WO 98/18501, WO98/05364, WO 98/17324.

In certain circumstances it can be desirable to include a precursor to agaseous substance (e.g., a material that is capable of being convertedto a gas in vivo, often referred to as a “gas precursor”). Preferablythe gas precursor and the gas it produces are physiologicallyacceptable. The gas precursor can be pH-activated, photo-activated,temperature activated, etc. For example, certain perfluorocarbons can beused as temperature activated gas precursors. These perfluorocarbons,such as perfluoropentane, have a liquid/gas phase transition temperatureabove room temperature (or the temperature at which the agents areproduced and/or stored) but below body temperature; thus they undergo aphase shift and are converted to a gas within the human body.

The gas can comprise a mixture of gases. The following combinations areparticularly preferred gas mixtures: a mixture of gases (A) and (B) inwhich, at least one of the gases (B), present in an amount of between0.5-41% by vol., has a molecular weight greater than 80 daltons and is afluorinated gas and (A) is selected from the group consisting of air,oxygen, nitrogen, carbon dioxide and mixtures thereof, the balance ofthe mixture being gas A.

Since ultrasound vesicles can be larger than the other detectable labelsdescribed herein, they can be linked or conjugated to a plurality ofcMet binding polypeptides or multimeric polypeptide constructs in orderto increase the targeting efficiency of the agent. Attachment to theultrasound contrast agents described above (or known to those skilled inthe art) can be via direct covalent bond between the cMet bindingpolypeptide and the material used to make the vesicle or via a linker,as described previously. For example, see WO 98/53857 generally for adescription of the attachment of a peptide to a bifunctional PEG linker,which is then reacted with a liposome composition (Lanza, G. et al.,1997. Ultrasound Med. Biol., 23:863-870).). The structure of thesecompounds typically comprises:

-   a) A hydrophobic portion, compatible with the material forming the    envelope of the microbubble or of the microballoon, in order to    allow an effective incorporation of the compound in the envelope of    the vesicle; said portion is typically a lipid moiety (e.g.,    dipalmitin, distearoil);-   b) A spacer (typically PEGs of different molecular weights), which    can be optional in some cases (microbubbles may, for instance, prove    difficult to freeze dry if the spacer is too long) or preferred in    some others (e.g., peptides can be less active when conjugated to a    microballoon with a short spacer);-   c) A reactive group capable of reacting with a corresponding    reactive moiety on the peptide to be conjugated (e.g., maleimido    with the —SH group of cysteine).

A number of methods can be used to prepare suspensions of microbubblesconjugated to cMet binding polypeptides. For example, one can preparemaleimide-derivatized microbubbles by incorporating 5% (w/w) of N-MPB-PE(1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-4-(p-maleimido-phenylbutyramide), (Avanti Polar-Lipids, Inc., Alabaster, Ala.) in thephospholipid formulation. Then, solutions of mercaptoacetylatedcMet-binding peptides (10 mg/mL in DMF), which have been incubated indeacetylation solution (50 mM sodium phosphate, 25 mM EDTA, 0.5 Mhydroxylamine.HCl, pH 7.5) are added to the maleimide-activatedmicrobubble suspension. After incubation in the dark, under gentleagitation, the peptide conjugated microbubbles can be purified bycentrifugation.

Alternatively, cMet-binding polypeptide conjugated microbubbles can beprepared using biotin/avidin. For example, avidin-conjugatedmicrobubbles can be prepared using a maleimide-activated phospholipidmicrobubble suspension, prepared as described above, which is added tomercaptoacetylated-avidin (which has been incubated with deacetylationsolution). Biotinylated cMet-binding peptides (prepared as describedherein) are then added to the suspension of avidin-conjugatedmicrobubbles, yielding a suspension of microbubbles conjugated tocMet-binding peptides.

Ultrasound imaging techniques, which can be used in accordance with thepresent invention, include known techniques, such as color Doppler,power Doppler, Doppler amplitude, stimulated acoustic imaging, and two-or three-dimensional imaging techniques. Imaging may be done in harmonic(resonant frequency) or fundamental modes, with the second harmonicpreferred.

C. Optical Imaging, Sonoluminescence or Photoacoustic Imaging

In accordance with the present invention, a number of optical parameterscan be employed to determine the location of cMet or HGF/cMet complexwith in vivo light imaging after injection of the subject with anoptically-labeled cMet binding polypeptides. Optical parameters to bedetected in the preparation of an image may include transmittedradiation, absorption, fluorescent or phosphorescent emission, lightreflection, changes in absorbance amplitude or maxima, and elasticallyscattered radiation. For example, biological tissue is relativelytranslucent to light in the near infrared (NIR) wavelength range of650-1000 nm. NIR radiation can penetrate tissue up to severalcentimeters, permitting the use of the cMet binding polypeptides ormultimeric polypeptide constructs of the present invention for opticalimaging of cMet or HGF/cMet complex in vivo.

The cMet binding polypeptides or multimeric polypeptide constructs canbe conjugated with photolabels, such as, for example, optical dyes,including organic chromophores or fluorophores, having extensivedelocalized ring systems and having absorption or emission maxima in therange of 400-1500 nm. The cMet binding polypeptide or multimericpolypeptide construct can alternatively be derivatized with abioluminescent molecule. The preferred range of absorption maxima forphotolabels is between 600 and 1000 nm to minimize interference with thesignal from hemoglobin. Preferably, photoabsorption labels have largemolar absorptivities, e.g., greater than 10⁵ cm⁻¹M⁻¹, while fluorescentoptical dyes will have high quantum yields. Examples of optical dyesinclude, but are not limited to those described in WO 98/18497, WO98/18496, WO 98/18495, WO 98/18498, WO 98/53857, WO 96/17628, WO97/18841, WO 96/23524, WO 98/47538, and references cited therein. Thephotolabels can be covalently linked directly to the cMet bindingpeptide or linked to the cMet binding peptide or multimeric polypeptideconstruct via a linker, as described previously.

After injection of the optically-labeled cMet binding moiety, thepatient is scanned with one or more light sources (e.g., a laser) in thewavelength range appropriate for the photolabel employed in the agent.The light used can be monochromatic or polychromatic and continuous orpulsed. Transmitted, scattered, or reflected light is detected via aphotodetector tuned to one or multiple wavelengths to determine thelocation of cMet or HGF/cMet complex in the subject. Changes in theoptical parameter can be monitored over time to detect accumulation ofthe optically-labeled reagent at the site of hyperproliferation.Standard image processing and detecting devices can be used inconjunction with the optical imaging reagents of the present invention.

The optical imaging reagents described above also can be used foracousto-optical or sonoluminescent imaging performed withoptically-labeled imaging agents (see, U.S. Pat. No. 5,171,298, WO98/57666, and references cited therein). In acousto-optical imaging,ultrasound radiation is applied to the subject and affects the opticalparameters of the transmitted, emitted, or reflected light. Insonoluminescent imaging, the applied ultrasound actually generates thelight detected. Suitable imaging methods using such techniques aredescribed in WO 98/57666.

D. Nuclear Imaging (Radionuclide Imaging) and Radiotherapy

The cMet binding moieties can be conjugated with a radionuclide reporterappropriate for scintigraphy, SPECT, or PET imaging and/or with aradionuclide appropriate for radiotherapy. Constructs in which the cMetbinding moieties are conjugated with both a chelator for a radionuclideuseful for diagnostic imaging and a chelator useful for radiotherapy arewithin the scope of the invention.

For use as a PET agent a peptide or multimeric polypeptide construct iscomplexed with one of the various positron emitting metal ions, such as⁵¹Mn, ⁵²Fe, ⁶⁰Cu, ⁶⁸Ga, ⁷²As, ⁹⁴ mTc, or ¹¹⁰In. The binding moieties ofthe invention can also be labeled by halogenation using radionuclidessuch as ¹⁸F, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹²³I, ⁷⁷Br, and ⁷⁶Br. Preferred metalradionuclides for scintigraphy or radiotherapy include ^(99m)Tc, ⁵¹ Cr,⁶⁷Ga, ⁶⁸Ga ⁴⁷Sc, ⁵¹Cr, ¹⁶⁷Tm, ¹⁴¹Ce, ¹¹¹In, ¹⁶⁸Yb, ¹⁷⁵Yb, ¹⁴⁰La, ⁹⁰Y,⁸⁸Y, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁶⁵Dy, ¹⁶⁶Dy, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁹⁷Ru, ¹⁸⁶Re, ¹⁸⁸Re,²⁰³Pb, ²¹¹Bi, ²¹²Bi, ²¹³Bi, ²¹⁴Bi, ¹⁶⁵Dy, ¹⁶⁶Dy, ⁶²Cu, ⁹⁷Ru, ¹⁰³Ru,¹⁸⁶Re, ¹⁸⁸Re, ²⁰³Pb, ²¹¹Bi, ²¹²Bi, ²¹³Bi, ²¹⁴Bi, ¹⁰⁵Rh, ¹⁰⁹Pd,^(117m)Sn, ¹⁴⁹Pm, ¹⁶¹Tb, ¹⁷⁷Lu, ¹⁹⁸Au and ¹⁹⁹Au. The choice of metalwill be determined based on the desired therapeutic or diagnosticapplication. For example, for diagnostic purposes the preferredradionuclides include ⁶⁴Cu, ⁶⁷Ga, ⁶⁸Ga, ^(99m)Tc, and ¹¹¹In. Fortherapeutic purposes, the preferred radionuclides include ⁶⁴Cu, ⁹⁰Y,¹⁰⁵Rh, ¹¹¹In, ^(117m)Sn, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁷⁵Yb,¹⁷⁷Lu, ^(186/188)Re, and ¹⁹⁹Au. ^(99m)Tc is particularly preferred fordiagnostic applications because of its low cost, availability, imagingproperties, and high specific activity. The nuclear and radioactiveproperties of ^(99m)Tc make this isotope an ideal scintigraphic imagingagent. This isotope has a single photon energy of 140 keV and aradioactive half-life of about 6 hours, and is readily available from a⁹⁹Mo-^(99m)Tc generator.

The metal radionuclides may be chelated by, for example, linear,macrocyclic, terpyridine, and N₃S, N₂S₂, or N₄ chelants (see also, U.S.Pat. No. 5,367,080, U.S. Pat. No. 5,364,613, U.S. Pat. No. 5,021,556,U.S. Pat. No. 5,075,099, U.S. Pat. No. 5,886,142), and other chelatorsknown in the art including, but not limited to, HYNIC, DTPA, EDTA, DOTA,DO3A, TETA, and bisamino bisthiol (BAT) chelators (see also U.S. Pat.No. 5,720,934). For example, N₄ chelators are described in U.S. Pat. No.6,143,274; U.S. Pat. No. 6,093,382; U.S. Pat. No. 5,608,110; U.S. Pat.No. 5,665,329; U.S. Pat. No. 5,656,254; and U.S. Pat. No. 5,688,487.Certain N₃S chelators are described in PCT/CA94/00395, PCT/CA94/00479,PCT/CA95/00249 and in U.S. Pat. No. 5,662,885; U.S. Pat. No. 5,976,495;and U.S. Pat. No. 5,780,006. The chelator also can include derivativesof the chelating ligand mercapto-acetyl-acetyl-glycyl-glycine (MAG3),which contains an N₃S, and N₂S₂ systems such as MAMA(monoamidemonoaminedithiols), DADS (N₂S diaminedithiols), CODADS and thelike. These ligand systems and a variety of others are described in, forexample, Liu, S, and Edwards, D., 1999. Chem. Rev., 99:2235-2268, andreferences therein.

The chelator also can include complexes containing ligand atoms that arenot donated to the metal in a tetradentate array. These include theboronic acid adducts of technetium and rhenium dioximes, such as aredescribed in U.S. Pat. No. 5,183,653; U.S. Pat. No. 5,387,409; and U.S.Pat. No. 5,118,797, the disclosures of which are incorporated byreference herein, in their entirety.

In another embodiment, disulfide bonds of a cMet binding polypeptide ofthe invention are used as two ligands for chelation of a radionuclidesuch as ^(99m)Tc. In this way the peptide loop is expanded by theintroduction of Tc (peptide-S—S-peptide changed topeptide-S—Tc—S-peptide). This also has been used in other disulfidecontaining peptides in the literature (Chen, J. et al., 2001. J. Nucl.Med., 42:1847-1855) while maintaining biological activity. The otherchelating groups for Tc can be supplied by amide nitrogens of thebackbone, another cystine amino acid or other modifications of aminoacids.

Particularly preferred metal chelators include those of Formula 20, 21,22, 23a, 23b, 24a, 24b and 25, set forth in FIGS. 8A-8F. Formulae 20-22are particularly useful for lanthanides such as paramagnetic Gd³⁺ andradioactive lanthanides such as ¹⁷⁷Lu, ⁹⁰Y, ¹⁵³Sm, ¹¹¹In, or ¹⁶⁶Ho.Formulae 23a-24b are particularly useful for radionuclides ^(99m)Tc,¹⁸⁶Re, or ¹⁸⁸Re. Formula 25 is particularly useful for ^(99m)Tc. Theseand other metal chelating groups are described in U.S. Pat. No.6,093,382 and U.S. Pat. No. 5,608,110, which are incorporated byreference herein in their entirety. Additionally, the chelating group offormula 22 is described in, for example, U.S. Pat. No. 6,143,274; thechelating group of formula 24 is described in, for example, U.S. Pat.No. 5,627,286 and U.S. Pat. No. 6,093,382, and the chelating group offormula 25 is described in, for example, U.S. Pat. No. 5,662,885; U.S.Pat. No. 5,780,006; and U.S. Pat. No. 5,976,495.

For formulae 24a and 24b of FIG. 8E, X is either CH₂ or O; Y is C₁-C₁₀branched or unbranched alky, aryl, aryloxy, arylamino, arylaminoacyl, orarylalkyl comprising C₁-C₁₀ branched or unbranched alkyl groups, hydroxyor C₁-C₁₀ branched or unbranched polyhydroxyalkyl groups, C₁-C₁₀branched or unbranched hydroxy or polyalkoxyalkyl orpolyhydroxy-polyalkoxyalkyl groups; J is C(═O)-, OC(═O)-, SO₂-, NC(═O)-,NC(═S)-, N(Y), NC(═NCH₃)-, NC(═NH)-, N═N-, homopolyamides orheteropolyamines derived from synthetic or naturally occurring aminoacids; and n is 1-100. Other variants of these structures are described,for example, in U.S. Pat. No. 6,093,382. The disclosures of each of theforegoing patents, applications and references are incorporated byreference herein, in their entirety.

The chelators can be covalently linked directly to the cMet bindingmoiety or multimeric polypeptide construct or linked to the cMet bindingpolypeptide via a linker, as described previously, and then directlylabeled with the radioactive metal of choice (see, WO 98/52618, U.S.Pat. No. 5,879,658, and U.S. Pat. No. 5,849,261).

Complexes of radioactive technetium are particularly useful fordiagnostic imaging and complexes of radioactive rhenium are particularlyuseful for radiotherapy. In forming a complex of radioactive technetiumwith the reagents of this invention, the technetium complex, preferablya salt of ^(99m)Tc pertechnetate, is reacted with the reagent in thepresence of a reducing agent. Preferred reducing agents are dithionite,stannous and ferrous ions; the most preferred reducing agent is stannouschloride. Means for preparing such complexes are conveniently providedin a kit form comprising a sealed vial containing a predeterminedquantity of a reagent of the invention to be labeled and a sufficientamount of reducing agent to label the reagent with ^(99m)Tc.Alternatively, the complex can be formed by reacting a peptide of thisinvention conjugated with an appropriate chelator with a pre-formedlabile complex of technetium and another compound known as a transferligand. This process is known as ligand exchange and is well known tothose skilled in the art. The labile complex can be formed using suchtransfer ligands as tartrate, citrate, gluconate or mannitol, forexample. Among the ^(99m)Tc pertechnetate salts useful with the presentinvention are included the alkali metal salts such as the sodium salt,or ammonium salts or lower alkyl ammonium salts.

Preparation of the Complexes of the Present Invention where the Metal isradioactive rhenium can be accomplished using rhenium starting materialsin the +5 or +7 oxidation state. Examples of compounds in which rheniumis in the Re(V11) state are NH₄ReO₄ or KReO₄. Re(V) is available as, forexample, [ReOCl₄](NBu₄), [ReOCl₄](AsPh₄), ReOCl₃(PPh₃)₂ and asReO₂(pyridine)⁴⁺, where Ph is phenyl and Bu is n-butyl. Other rheniumreagents capable of forming a rhenium complex also can be used.

Radioactively labeled scintigraphic imaging agents provided by thepresent invention are encompassed having a suitable amount ofradioactivity. Generally, the unit dose to be administered has aradioactivity of about 0.01 mCi to about 100 mCi, preferably 1 mCi to 20mCi. The solution to be injected at unit dosage is from about 0.01 mL toabout 10 mL. In forming ^(99m)Tc radioactive complexes, it is generallypreferred to form radioactive complexes in solutions containingradioactivity at concentrations of from about 0.01 mCi to 100 mCi permL.

Typical doses of a radionuclide-labeled cMet binding imaging agentsaccording to the invention provide 10-20 mCi. After injection of thecMet-specific radionuclide imaging agent into the patient, a gammacamera calibrated for the gamma ray energy of the nuclide incorporatedin the imaging agent is used to image areas of uptake of the agent andquantify the amount of radioactivity present in the site. Imaging of thesite in vivo can take place in a matter of a few minutes. However,imaging can take place, if desired, in hours or even longer, after theradiolabeled peptide is injected into a patient. In most instances, asufficient amount of the administered dose will accumulate in the areato be imaged within about 0.1 of an hour to permit the taking ofscintiphotos.

Proper dose schedules for the radiotherapeutic compounds of the presentinvention are known to those skilled in the art. The compounds can beadministered using many methods including, but not limited to, a singleor multiple IV or IP injections, using a quantity of radioactivity thatis sufficient to cause damage or ablation of the targetedcMet-expressing tissue, but not so much that substantive damage iscaused to non-target (normal tissue). The quantity and dose required isdifferent for different constructs, depending on the energy andhalf-life of the isotope used, the degree of uptake and clearance of theagent from the body and the mass of the tumor. In general, doses canrange from a single dose of about 30-50 mCi to a cumulative dose of upto about 3 Ci.

The radiotherapeutic compositions of the invention can includephysiologically acceptable buffers, and can require radiationstabilizers to prevent radiolytic damage to the compound prior toinjection. Radiation stabilizers are known to those skilled in the art,and can include, for example, para-aminobenzoic acid, ascorbic acid,gentistic acid and the like.

A single, or multi-vial kit that contains all of the components neededto prepare the complexes of this invention, other than the radionuclide,is an integral part of this invention.

A single-vial kit preferably contains a chelating ligand, a source ofstannous salt, or other pharmaceutically acceptable reducing agent, andis appropriately buffered with pharmaceutically acceptable acid or baseto adjust the pH to a value of about 3 to about 9. The quantity and typeof reducing agent used would depend on the nature of the exchangecomplex to be formed. The proper conditions are well known to those thatare skilled in the art. It is preferred that the kit contents be inlyophilized form. Such a single vial kit can optionally contain labileor exchange ligands such as glucoheptonate, gluconate, mannitol, malate,citric or tartaric acid and can also contain reaction modifiers such asdiethylenetriamine-pentaacetic acid (DPTA), ethylenediamine tetraaceticacid (EDTA), or α, β, or γ cyclodextrin that serve to improve theradiochemical purity and stability of the final product. The kit alsocan contain stabilizers, bullring agents such as mannitol, that aredesigned to aid in the freeze-drying process, and other additives knownto those skilled in the art.

A multi-vial kit preferably contains the same general components butemploys more than one vial in reconstituting the radiopharmaceutical.For example, one vial can contain all of the ingredients that arerequired to form a labile Tc(V) complex on addition of pertechnetate(e.g., the stannous source or other reducing agent). Pertechnetate isadded to this vial, and after waiting an appropriate period of time, thecontents of this vial are added to a second vial that contains theligand, as well as buffers appropriate to adjust the pH to its optimalvalue. After a reaction time of about 5 to 60 minutes, the complexes ofthe present invention are formed. It is advantageous that the contentsof both vials of this multi-vial kit be lyophilized. As above, reactionmodifiers, exchange ligands, stabilizers, bulking agents, etc. can bepresent in either or both vials.

Therapeutic Applications

The cMet binding polypeptides and multimeric polypeptide constructs ofthe present invention can be used to present, treat or improve theactivity of therapeutic agents such as anti-proliferative or tumoricidalagents against undesired cellular proliferation (such as occurs inneoplastic tumors, e.g., cancer, by providing or improving theiraffinity for cMet and their residence time at a HGF/cMet complex onproliferating cells, such as, for example, epithelial cells) fordiseases associated with cMet, including, but not limited to, diseasesrelated to cMet activity. In this aspect of the invention, hybrid agentsare provided by conjugating a cMet binding polypeptide or multimericpolypeptide construct according to the invention with a therapeuticagent. The therapeutic agent can be a radiotherapeutic, discussed above,a drug, chemotherapeutic or tumoricidal agent, genetic material or agene delivery vehicle, etc. The cMet binding polypeptide moiety portionof the conjugate causes the therapeutic to “home” to the sites of cMetor HGF/cMet complex (i.e., activated epithelial cells), and to improvethe affinity of the conjugate for the endothelium, so that thetherapeutic activity of the conjugate is more localized and concentratedat the sites of cellular proliferation. In addition, these cMet bindingmoieties can inhibit HGF-mediated signaling events by preventing HGFfrom binding to cMet. Such conjugates will be useful in treatinghyperproliferative disorders, especially neoplastic tumor growth andmetastasis, in mammals, including humans. The method comprisesadministering to a mammal in need thereof an effective amount of a cMetbinding polypeptide or multimeric polypeptide construct according to theinvention conjugated with a therapeutic agent. The invention alsoprovides the use of such conjugates in the manufacture of a medicamentfor the treatment of angiogenesis associated diseases in mammals,including humans.

Suitable therapeutic agents for use in this aspect of the inventioninclude, but are not limited to: antineoplastic agents, such as platinumcompounds (e.g., spiroplatin, cisplatin, and carboplatin), methotrexate,adriamycin, mitomycin, ansamitocin, bleomycin, cytosine, arabinoside,arabinosyl adenine, mercaptopolylysine, vincristine, busulfan,chlorambucil, melphalan (e.g., PAM, L-PAM, or phenylalanine mustard),mercaptopurine, mitotane, procarbazine hydrochloride, dactinomycin(actinomycin D), daunorubcin hydrochloride, doxorubicin hydrochloride,taxol, mitomycin, plicamycin (mithramycin), aminoglutethimide,estramustine phosphate sodium, flutamide, leuprolide acetate, megestrolacetate, tamoxifen citrate, testoiactone, trilostane, amsacrine(m-AMSA), aparaginase (L-aparaginase), Erwina aparaginase, etoposide(VP-16), interferon CX-2a, Interferon CX-2b, teniposide (VM-26,vinblastine sulfate (VLB), vincristine sulfate, bleomycin sulfate,adriamycin, and arabinosyl; anti-angiogenic agents such as tyrosinekinase inhibitors with activity toward signaling molecules important inangiogenesis and/or tumor growth such as SU5416 and SU6668(Sugen/Pharmacia and Upjohn), endostatin (EntreMed), angiostatin(EntreMed), Combrestatin (Oxigene), cyclosporine, 5-fluorouracil,vinblastine, doxorubicin, paclitaxel, daunorubcin, immunotoxins;coagulation factors; antivirals such as acyclovir, amantadineazidothymidine (AZT or Zidovudine), ribavirin and vidarabine monohydrate(adenine arahinoside, ara-A); antibiotics, antimalarials, antiprotozoanssuch as chloroquine, hydroxychloroquine, metroidazole, quinine andmeglumine antimonate; anti-inflammatories such as diflunisal, ibuprofen,indomethacin, meclofenamate, mefenamic acid, naproxen, oxyphenbutazone,phenylbutazone, piroxicam, sulindac, tolmetin, aspirin and salicylates.

In one embodiment of the invention, the therapeutic agent can beassociated with an ultrasound contrast agent composition in which cMetbinding moieties of the invention are linked to the material employed toform the vesicles as described herein. After administration of theultrasound contrast agent and the optional imaging of the contrast agentbound to the tissue expressing cMet or HGF/cMet complex, the tissue canbe irradiated with an energy beam (preferably ultrasonic, e.g., with afrequency of from 0.3 to 3 MHz), to rupture or burst the microvesicles.The therapeutic effect of the therapeutic agent can thus be enhanced bythe energy released by the rupture of the microvesicles, in particularcausing an effective delivery of the therapeutic agent to the targetedtissue. For instance, the therapeutic agent can be associated with thetargeted ultrasound contrast agent and delivered as described in U.S.Pat. No. 6,258,378, herein incorporated by reference.

The cMet binding polypeptides and multimeric polypeptide constructs ofthe present invention also can be used to target genetic material tocMet-expressing cells. Thus, they can be useful in gene therapy,particularly for treatment of hyperproliferative disorders. In thisembodiment, genetic material or one or more delivery vehicles containinggenetic material useful in treating a hyperproliferative disorder can beconjugated to one or more cMet binding moieties of the invention andadministered to a patient. The genetic material can include nucleicacids, such as RNA or DNA, of either natural or synthetic origin,including recombinant RNA and DNA and antisense RNA and DNA. Types ofgenetic material that can be used include, for example, genes carried onexpression vectors such as plasmids, phagemids, cosmids, yeastartificial chromosomes (YACs) and defective or “helper” viruses,antigene nucleic acids, both single and double stranded RNA and DNA andanalogs thereof, such as phosphorothioate and phosphorodithioateoligodeoxynucleotides. Additionally, the genetic material can becombined, for example, with lipids, proteins or other polymers. Deliveryvehicles for genetic material can include, for example, a virusparticle, a retroviral or other gene therapy vector, a liposome, acomplex of lipids (especially cationic lipids) and genetic material, acomplex of dextran derivatives and genetic material, etc.

In a preferred embodiment the constructs of the invention are utilizedin gene therapy for treatment of hyperproliferative disorders. In thisembodiment, genetic material, or one or more delivery vehiclescontaining genetic material, e.g., useful in treating ahyperproliferative disorder, can be conjugated to one or more cMetbinding polypeptides or multimeric polypeptide constructs of theinvention and administered to a patient.

Constructs including genetic material and the cMet-binding moieties ofthe invention can be used, in particular, to selectively introduce genesinto proliferating cancer cells (e.g., epithelial cells), which can beuseful to treat cancer.

Therapeutic agents and the cMet binding moieties of the invention can belinked or fused in known ways, optionally using the same type of linkersdiscussed elsewhere in this application. Preferred linkers will besubstituted or unsubstituted alkyl chains, amino acid chains,polyethylene glycol chains, and other simple polymeric linkers known inthe art. More preferably, if the therapeutic agent is itself a protein,for which the encoding DNA sequence is known, the therapeutic proteinand cMet binding polypeptide can be coexpressed from the same syntheticgene, created using recombinant DNA techniques, as described above. Thecoding sequence for the cMet binding polypeptide can be fused in framewith that of the therapeutic protein, such that the peptide is expressedat the amino- or carboxy-terminus of the therapeutic protein, or at aplace between the termini, if it is determined that such placement wouldnot destroy the required biological function of either the therapeuticprotein or the cMet binding polypeptide. A particular advantage of thisgeneral approach is that concatamerization of multiple, tandemlyarranged cMet binding polypeptides is possible, thereby increasing thenumber and concentration of cMet binding sites associated with eachtherapeutic protein. In this manner cMet binding avidity is increased,which would be expected to improve the efficacy of the recombinanttherapeutic fusion protein.

Additionally, constructs including cMet binding polypeptides of thepresent invention can themselves be used as therapeutics to treat anumber of diseases associated with cMet activity. For example, wherebinding of a protein or other molecule (e.g., a growth factor, hormoneetc.) is necessary for or contributes to a disease process and a bindingmoiety inhibits such binding, constructs including such binding moietiescould be useful as therapeutics. Similarly, where binding of a bindingmoiety itself inhibits a disease process, constructs containing suchbinding moieties also could be useful as therapeutics.

The binding of HGF to cMet results in the activation of numerousintracellular signal transduction pathways leading to hyperproliferationof various cells. As such, in one embodiment, constructs including cMetbinding polypeptides that inhibit the binding of HGF to cMet (orotherwise inhibit activation of cMet) can be used as anti-neoplasticagents. In addition, as binding of HGF and activation of cMet isimplicated in angiogenic activity, in another embodiment, constructsincluding cMet binding polypeptides that inhibit the binding of HGF tocMet, or otherwise inhibit activation of cMet, can be used asanti-angiogenic agents. Certain constructs of the invention includingmonomers, multimers and heteromultimers that inhibit activation of cMetare also discussed in the Examples, and include, for example, SEQ IDNO:365 (FIG. 10). The binding polypeptides and constructs thereof of thepresent invention are useful as therapeutic agents for treatingconditions that involve endothelial and/or epithelial cells expressingcMet. Because an important function of endothelium is angiogenesis, orthe formation of blood vessels, the polypeptides and constructs thereofare particularly useful for treating conditions that involveangiogenesis and/or hyperproliferation. Conditions that involveangiogenesis include, for example, solid tumors, tumor metastases andbenign tumors. Tumors caused by cMet activation or through angiogenesisare well known in the art and include, for example, breast, thyroid,glioblastoma, prostate, malignant mesothelioma, colorectal,hepatocellular, hepatobiliary, renal, osteosarcoma and cervical.Additional tumors and related disorders are listed in Table I of U.S.Pat. No. 6,025,331, issued Feb. 15, 2000 to Moses, et al., the teachingsof which are incorporated herein by reference. Benign tumors include,for example, hemangiomas, acoustic neuromas, neurofibromas, trachomas,and pyogenic granulomas. Other relevant diseases that involveangiogenesis and/or hyperproliferation include for example, rheumatoidarthritis, psoriasis, and ocular diseases, such as diabetic retinopathy,retinopathy of prematurity, macular degeneration, corneal graftrejection, neovascular glaucoma, retrolental fibroplasia, rebeosis,Osler-Webber Syndrome, myocardial angiogenesis, plaqueneovascularization, telangiectasia, hemophiliac joints, angiofibroma andwound granulation. Other relevant diseases or conditions that involveblood vessel growth include intestinal adhesions, atherosclerosis,scleroderma, and hypertropic scars, and ulcers. Furthermore, the bindingpolypeptides and constructs thereof of the present invention can be usedto reduce or prevent uterine neovascularization required for embryoimplantation, for example, as a birth control agent.

The binding polypeptides, multimeric polypeptide constructs andconstructs conjugates thereof can be administered to an individual overa suitable time course depending on the nature of the condition and thedesired outcome. They binding, polypeptides and constructs thereof canbe administered prophylactically, e.g., before the condition isdiagnosed or to an individual predisposed to a condition. The bindingpolypeptides multimeric polypeptide constructs and conjugates andconstructs thereof can be administered while the individual exhibitssymptoms of the condition or after the symptoms have passed or otherwisebeen relieved (such as after removal of a tumor). In addition, theybinding polypeptides and constructs thereof of the present invention canbe administered a part of a maintenance regimen, for example to preventor lessen the recurrence or the symptoms or condition. As describedbelow, the binding polypeptides multimeric polypeptide constructs andconjugates and constructs thereof of the present invention can beadministered systemically or locally.

The quantity of material administered will depend on the seriousness ofthe condition. For example, for treatment of a hyperproliferativedisorder, e.g., in the case of neoplastic tumor growth, the position andsize of the tumor will affect the quantity of material to beadministered. The precise dose to be employed and mode of administrationmust per force, in view of the nature of the complaint, be decidedaccording to the circumstances by the physician supervising treatment.In general, dosages of the agent conjugate polypeptides, multimericpolypeptide constructs and conjugates of the present invention willfollow the dosages that are routine for the therapeutic agent alone,although the improved affinity of a binding polypeptide or multimericpolypeptide construct of the invention for its target can allow for adecrease in the standard dosage.

Such conjugate pharmaceutical compositions are preferably formulated forparenteral administration, and most preferably for intravenous orintra-arterial administration. Generally, and particularly whenadministration is intravenous or intra-arterial, pharmaceuticalcompositions can be given as a bolus, as two or more doses separated intime, or as a constant or non-linear flow infusion.

As used herein the term “therapeutic” includes at least partialalleviation of symptoms of a given condition. The binding polypeptides,multimeric constructs and constructs conjugates thereof of the presentinvention do not have to produce a complete alleviation of symptoms tobe useful. For example, treatment of an individual can result in adecrease in the size of a tumor or diseased area, or prevention of anincrease in size of the tumor or diseased area. Treatment also canprevent or lessen the number or size of metastatic outgrowths of themain tumor(s).

Symptoms that can be alleviated include physiological characteristicssuch as cMet activity. The binding polypeptides multimeric polypeptideconstructs and conjugates and constructs thereof of the presentinvention can inhibit activity of cMet and its homologs by binding tocMet and inhibiting its activity or by binding to cMet and inhibitingHGF from activating this receptor. Such inhibition can be detected, forexample, by measuring the phosphorylation state of the receptor in thepresence of or after treatment with the binding polypeptides orconstructs thereof. Based on the teachings provided herein, one ofordinary skill in the art would know how and be able to administer asuitable dose of binding polypeptide, multimeric polypeptide constructsand conjugates or construct thereof as provided herein, and measure theeffect of treatment on the parameter of interest. For example, the sizeof the area of interest (e.g., the tumor or lesion) can be measuredbefore and after treatment. Cells or cMet itself can be isolated fromthe sample and used in assays described herein.

The dosage of the polypeptides multimeric polypeptide constructs andconjugates and constructs thereof can depend on the age, sex, health,and weight of the individual, as well as the nature of the condition andoverall treatment regimen. The biological effects of the polypeptidesmultimeric polypeptide constructs and conjugates and constructs thereofare described herein. Therefore, based on the biological effects of thebinding polypeptides multimeric polypeptide constructs and conjugatesand constructs provided herein, and the desired outcome of treatment,the preferred dosage is determinable by one of ordinary skill in the artthrough routine optimization procedures. Typically, the daily regimen isin the range of about 0.1 mg/kg to about 1 mg/kg.

The binding polypeptides moieties and constructs conjugates thereofprovided herein can be administered as the sole active ingredient,optionally together with a pharmaceutically acceptable excipient, or canbe administered together (e.g., simultaneously or sequentially) withother binding polypeptides and constructs thereof, other therapeuticagents, or combination thereof. In addition, the binding polypeptidesmoieties and conjugate constructs thereof can be conjugated totherapeutic agents, for example, to improve specificity, residence timein the body, or therapeutic effect. Such othertherapeutic agentsinclude, for example, other anti-proliferative compounds, andtumoricidal compounds. The therapeutic agent also can includeantibodies. Furthermore, the binding polypeptide multimeric polypeptideconstructs and constructs thereof of the present invention can be usedas a cancer cell homing device. Therefore, they binding polypeptide orconstructs thereof can may be conjugated to nucleic acid encoding, forexample, a therapeutic polypeptide, in order to target the nucleic acidto stromal cells. Once exposed to the nucleic acid conjugated bindingpolypeptide moiety or conjugate thereof, the stromal cells caninternalize and express the conjugated nucleic acid, thereby deliveringthe therapeutic peptide to the target cells.

The binding polypeptides, multimeric polypeptide constructs andconjugates and constructs thereof can be administered locally orsystemically by any suitable route. Suitable routes of administrationinclude, but are not limited to, topical application, transdermal,parenteral, gastrointestinal, intravaginal, and transalveolar.Compositions for the desired route of administration can be prepared byany of the methods well known in the pharmaceutical arts, for example,as described in Remington: The Science and Practice of Pharmacy, 20thed., Lippincott, Williams and Wilkins, 2000.

For topical application, the binding polypeptides, multimericpolypeptide constructs and conjugates thereof can be suspended, forexample, in a cream, gel or rinse that allows the polypeptides orconstructs to penetrate the skin and enter the blood stream, forsystemic delivery, or contact the area of interest, for localizeddelivery. Compositions suitable for topical application include anypharmaceutically acceptable base in which the polypeptides or constructsare at least minimally soluble.

For transdermal administration, the polypeptides, multimeric polypeptideconstructs and conjugates thereof can be applied in pharmaceuticallyacceptable suspension together with a suitable transdermal device or“patch”. Examples of suitable transdermal devices for administration ofthe polypeptides or constructs of the present invention are described,for example, in U.S. Pat. No. 6,165,458, issued Dec. 26, 2000 toFoldvari et al., and U.S. Pat. No. 6,274,166B1, issued Aug. 4, 2001 toSintov et al., the teachings of which are incorporated herein byreference.

For parenteral administration, the polypeptides, multimeric polypeptideconstructs and conjugates thereof can be injected intravenously,intramuscularly, intraperitoneally, or subcutaneously. Typically,compositions for intravenous administration are solutions in sterileisotonic aqueous buffer. Other pharmaceutically acceptable carriersinclude, but are not limited to, sterile water, saline solution, andbuffered saline (including buffers like phosphate or acetate), alcohol,vegetable oils, polyethylene glycols, gelatin, lactose, amylose,magnesium stearate, talc, silicic acid, paraffin, etc. Where necessary,the composition also can include a solubilizing agent and a localanaesthetic such as lidocaine to ease pain at the site of the injection,preservatives, stabilizers, wetting agents, emulsifiers, salts,lubricants, etc. as long as they do not react deleteriously with theactive compounds. Similarly, the composition may comprise conventionalexcipients, i.e. pharmaceutically acceptable organic or inorganiccarrier substances suitable for parenteral, enteral or intranasalapplication which do not deleteriously react with the active compounds.Generally, the ingredients will be supplied either separately or mixedtogether in unit dosage form, for example, as a dry lyophilized powderor water free concentrate in a hermetically sealed container such as anampoule or sachette indicating the quantity of active agent in activityunits. Where the composition is to be administered by infusion, it canbe dispensed with an infusion bottle containing sterile pharmaceuticalgrade “water for injection” or saline. Where the composition is to beadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients can be mixed prior toadministration.

For gastrointestinal and intravaginal administration, the polypeptides,multimeric polypeptide constructs and conjugates thereof can beincorporated into pharmaceutically acceptable powders, pills or liquids,and suppositories for rectal or vaginal administration.

For transalveolar, buccal or pulmonary administration, the polypeptides,multimeric polypeptide constructs and conjugates thereof can besuspended in a pharmaceutically acceptable excipient suitable foraerosolization and inhalation or as a mouthwash. Devices suitable fortransalveolar administration such as atomizers and vaporizers also areincluded within the scope of the invention. Suitable formulations foraerosol delivery of polypeptides, etc. using buccal or pulmonary routescan be found, for example in U.S. Pat. No. 6,312,665B1, issued Nov. 6,2001 to Pankaj Modi, the teachings of which are incorporated herein byreference.

In addition, the polypeptides, multimeric polypeptide constructs andconjugates thereof of the present invention can be administered nasallyor ocularly, where the polypeptide or construct is suspended in a liquidpharmaceutically acceptable agent suitable for drop-wise dosing.

The polypeptides, multimeric polypeptide constructs and conjugatesthereof of the present invention can be administered such that thepolypeptide, etc. is released in the individual over an extended periodof time (sustained or controlled release). For example, the polypeptide,multimeric polypeptide constructs and conjugates thereof can beformulated into a composition such that a single administration providesdelivery of the polypeptide, etc. for at least one week, or over theperiod of a year or more. Controlled release systems include monolithicor reservoir-type microcapsules, depot implants, osmotic pumps,vesicles, micelles, liposomes, transdermal patches and iontophoreticdevices. In one embodiment, the polypeptides, multimeric polypeptideconstructs and conjugates thereof of the present invention areencapsulated or admixed in a slowly degrading, non-toxic polymer.Additional formulations suitable for controlled release of thepolypeptides, multimeric polypeptide constructs and conjugates thereofprovided herein are described in U.S. Pat. No. 4,391,797, issued Jul. 5,1983, to Folkman et al., the teachings of which are incorporated hereinby reference.

Another suitable method for delivering the polypeptides of the presentto an individual is via in viva production of the polypeptide. A geneencoding the polypeptide can be administered to the individual such thatthe encoded polypeptide is expressed. The gene can be transientlyexpressed. In a particular embodiment, the gene encoding the polypeptideis transfected into cells that have been obtained from the patient, amethod referred to as ex vivo gene therapy. Cells expressing thepolypeptide are then returned to the patient's body. Methods of ex vivogene therapy are well known in the art and are described, for example,in U.S. Pat. No. 4,391,797, issued Mar. 21, 1998 to Anderson et al., theteachings of which are incorporated herein by reference.

Isolation of cMet binding moieties polypeptides and preparation and useof cMet binding moieties and conjugates thereof in accordance with thisinvention will be further illustrated in the following examples. Thespecific parameters included in the following examples are intended toillustrate the practice of the invention, and they are not presented toin any way limit the scope of the invention.

EXAMPLES Example 1 Method for Identification of cMet-BindingPolypeptides

A four-pronged selection strategy using a variety of peptide-displayingphage libraries was utilized to screen for cMet-binding polypeptides.Both the extracellular domain of the cMet receptor (expressed as anFc-fusion protein) and the colorectal cancer cell line, DLD-1, whichexpress high levels of cMet on their cell surface, were used as toolsfor the selections.

Briefly, the selections involved either using the soluble cMet-Fc-fusionprotein or DLD-1 cells as the target. Specific elutions with HGF (firstfor 1 hour and then overnight to identify both low and high affinitycMet binders) were performed. Additionally, while using the soluble cMetreceptor, all peptide-displaying phage that remained bound to thereceptor were harvested to identify peptides that did not bind to theligand binding site, but could nevertheless be potentially developedinto imaging agents. FIG. 9 illustrates the selection strategy that wasemployed. Briefly, 21 different selection campaign/elution combinationswere performed with each library pool. An additional 10 selectioncampaigns representing rounds 3 and 4 using the soluble Met-Fc fusionprotein were also performed. HGF elutions were at a concentration of 100ng/mL.

Example 2 Determination of Peptide-Display Phage Binding to SolublecMet-Fc Fusion Protein “Protein Phage Elisas”

Protein phage ELISAs using peptide-displaying phage isolates from thevarious selection campaigns were performed to determine specificity ofthe peptides for cMet versus an unrelated Fc-fusion protein (TRAIL-Fe).Briefly, 384-well plates were coated overnight at 4 C with 0.5 μg/mL ofcMet-Fc fusion protein or TRAIL-Fc fusion protein (background). Theplates were blocked for 2 hours 37 C with 3% (w/v) BSA in PBS containing0.05% (v/v) Tween-20 (PBST). The plates were washed with PBST and 100 μLof peptide-displaying phage were added to each well. The plates wereincubated for 2 hours at room temperature and washed with PBST.cMet-binding peptide-displaying phage were detected using anHRP-conjugated anti-M13 antibody.

The peptide-displaying phage that demonstrated a>3-fold binding tocMet-Fc fusion protein versus TRAIL-Fe fusion protein are hereinreferred to as “positive hits”. The positive hits identified in theabove screen were subjected to DNA sequencing. From subsequent sequenceanalysis, 187 unique peptide sequences were identified. Thecorresponding amino acid sequences of the cMet-binding phage-displayedpeptides are listed in Table 1 (SEQ ID NO: 001-101, 365-387, 390-404,449-496).

Example 3 Determination of cMet Binding in a Cellular Model

Whole cell ELISAs were performed to assess whether the positive hitsdemonstrated specific binding to cell surface-expressed human cMet.

Whole cell ELISAs were performed using 3T3 cells that over-express humancMet. 3T3 cells that do not express cMet (“non-expressing cells”) wereused as a control cell line. Briefly, 96-well plates were seeded with10⁵ cells per well. The plates were centrifuged for 5 minutes at 1600rpm to pellet the cells. The resulting cell layer was fixed with 0.1%(v/v) glutaraldehyde for 12 minutes at 37 C. The cells were washed withPBS and subsequently blocked with 3% BSA in PBST for 1 hour at 37 C.Peptide-displaying phage also were blocked in the above solution for 1hour at 37 C. 100 μL of blocked phage was then added to each well andthe plates were incubated for 1 hour at room temperature. The plateswere washed with PBST. cMet-binding peptide-displaying phage weredetected using an HRP-conjugated anti-M13 antibody.

Example 4 HGF Competition Protein ELISAs

HGF competition protein ELISAs were performed in an attempt to determinewhether any of the cMet-binding peptides compete with HGF for a similarbinding site on cMet. This competition ELISA identifies peptides thatserve as “HGF antagonistic peptides”, peptides that block HGF-mediatedsignaling events (e.g., proliferation). These assays were conductedusing the peptide-displaying phage discovered from the initial selectionand screening campaigns using the first generation peptide libraries.Briefly, 96-well plates were coated overnight at 4 C with 0.5 μg/mL ofcMet-Fc fusion protein or TRAIL-Fc fusion protein (background). Theplates were blocked for 2 hours at 37 C with 3% BSA in PBST. The plateswere washed with PBST, and 100 μL of HGF (either at 100 ng/mL or 500ng/mL in PBST) was added to each well. The plates were incubated for 30minutes at room temperature after which the plates were washed with PBSTand 70 μL of HGF (143 ng/mL or 714 ng/mL) or 70 mL of PBST was added tothe respective wells. This was followed by an addition of 30 μL ofpeptide-displaying phage overnight culture to each well. The plates wereincubated for 2 hours at room temperature, washed with PBST andcMet-binding peptide-displaying phage was detected using anHRP-conjugated anti-M13 antibody.

Data for the protein ELISAs, whole cell ELISAs and the HGF competitionexperiments is presented in Table 7.

Example 5 Peptide Synthesis and Fluorescein Labeling

A select number of cMet-binding peptides corresponding to positive phageisolates were synthesized on a solid phase matrix using9-fluorenylmethoxycarbonyl protocols. These peptides were purified withreverse phase chromatography. Peptide masses were confirmed byelectrospray mass spectrometry, and peptides were quantified bymeasuring absorbance at 280 nm. For synthesis, two N-terminal and twoC-terminal amino acids from the phage vector sequence from which thepeptide was excised were retained, and a linker, e.g.,-Gly-Gly-Gly-Lys-NH₂ (SEQ ID NO:513) was added to the C-terminus of eachpeptide. Each peptide was N-terminally acetylated. Selected lysineresidues were protected with1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde) whereappropriate. The protecting group allows for selective coupling to theC-terminal lysine, is not removed during peptide cleavage, but can beremoved after coupling with 2% hydrazine in DMF or 0.5 M hydroxylamine,pH 8, in water.

Each peptide was labeled with fluorescein on the C-terminal lysine usingfluorescein (N-hydroxysuccinimide ester derivative) or fluoresceinisothiocyanate (FITC) in DMF with 2% diisopropylethylamine (DIPEA). Inthe case where the peptide contained an ivDde protected lysine, thereaction was quenched by the addition of 2% hydrazine, which reacts withall free NHS-fluorescein and removes the internal protecting group. Forall other peptides, the reaction was quenched by the addition of anequal volume of 0.5M hydroxylamine, pH 8. The quenched reactions werethen diluted with water to less than 10% DMF and then purified using C18reverse phase chromatography. The peptides were verified by analyzingthem for expected mass using an LC-MS system (HP1100 HPLC with in-lineSCIEX AP150 single quadrapole mass spectrometer), and the purity of thepeptides was determined.

Example 6 Fluorescence Anisotropy Measurements

Fluorescence anisotropy measurements were performed in 384-wellmicroplates in a volume of 10 μL in binding buffer (PBS, 0.01% Tween-20,pH 7.5) using a Tecan Polarion fluorescence polarization plate reader(Caracas, Venezuela). The concentration of fluorescein-labeled peptidewas held constant (20 nM) and the concentration of cMet-Fc fusionprotein (or similar target) was varied. Binding mixtures wereequilibrated for 10 minutes in the microplate at 30 C beforemeasurement. The observed change in anisotropy was fit to the equationbelow via nonlinear regression to obtain the apparent K_(D). Thisequation (1) assumes that the synthetic peptide and cMet form areversible complex in solution with 1:1 stoichiometry.

$r_{obs} = {r_{free} + {\left( {r_{bound} - r_{free}} \right)\frac{\left( {K_{D} + {cMet} + P} \right) - \sqrt{{\left( {K_{D} + {cMet} + P} \right)2} - {4 \cdot {cMet} \cdot P}}}{2 \cdot P}}}$

where r_(obs) is the observed anisotropy, r_(free) is the anisotropy ofthe free peptide, r_(bound) is the anisotropy of the bound peptide,K_(D) is the apparent dissociation constant, cMet is the total cMetconcentration, and P is the total fluorescein-labeled peptideconcentration. K_(D) was calculated in a direct binding assay (K_(D,B))and therefore these values represent cMet binding to the fluoresceinlabeled peptide.

Example 7 Peptide Competition Fluorescence Polarization Assays

Peptide competition fluorescence polarization assays were performed todetermine which peptides compete with each other for binding to cMet.This would identify potential heteromeric peptide complexes that exhibithigher affinity for the cMet receptor than an individual peptide alone.

Briefly, cross competition of cMet-binding peptides was performed on aCartesian liquid handler (Irvine, Calif.) in a 3 μL total reactionvolume. Flourescein-labeled peptides were diluted to a finalconcentration of 20 nM and unlabeled competitor peptides were diluted toa final concentration of 10 μM. cMet-Fc fusion protein was diluted tothe K_(D) for each fluorescein-labeled peptide in the reaction. Bindingmixtures were equilibrated for 10 minutes on the microplate at 30 Cbefore measuring any changes in anisotropy. From these studies, threepairs of cMet-binding peptides were identified as being non-competitorsand represent ideal candidates for heteromeric cMet-binding peptidecomplexes (see Table 9).

Example 8 General Procedure for Preparation of Heteromeric cMet-BindingPeptide Complexes

Each of the dimers consists of a Tc-chelating 6-PnAO ligand bearingsequence (generally referred to as A) and a spacer functionalized(spacer=JJ; J=8-Amino-3,6-dioxaoctanoic acid) portion (genericallyreferred to as B). Compound B was treated with a 10-fold excess ofglutaric acid his NHS ester (Tyger Scientific, Princeton, N.J.) and˜20-fold excess of diisopropylethylamine at ambient temperature in DMFfor 30 minutes. The reaction mixture was diluted with ether (15-fold byvolume) which led to the precipitation of the mono-NHS ester of theglutarylated peptide. The ether was decanted and the solid washed thricemore with ether, which removed any traces of unreacted glutaric acid bisNHS ester. The resulting solid was resuspended in dry DMF and thecompound A (1 equiv) was added followed by diisopropylethylamine (20equiv) and the mixture was stirred for 24 hours at ambient temperature.The mixture was diluted with water (50-fold) and the mixture wasdirectly loaded onto a reverse-phase HPLC column, which was eluted witha gradient of acetonitrile (0.1% TFA) into water (0.1% TFA). Fractionscontaining the desired product were combined and lyophilized to providethe desired materials.

Specific Example Preparation of Heterodimeric cMet-Binding PeptidesComplexes 1) Preparation of a PnAOG-Glut Modified SEQ ID NO:514 Peptide(A Type A Compound)

To a solution of 6-Glutaryl-PnAO (40 mg, 0.1 mmol) in dry DMF (0.2 mL)was added N-hydroxysuccinimide (NHS, 14 mg, 0.12 mmol) anddiisopropylcarbodiimide (DIC, 15 mg, 0.12 mmol) and stirred for 4 h atroom temperature. Ether:hexane (5 mL, 1:1) was added to the reactionmixture. The mixture was stirred and the supernatant solution wasremoved by decantation, leaving behind the paste in the flask. The pastewas washed with ether:hexane (1:1) (3×5 mL) and dissolved in dry DMF(0.2 mL). To this solution were added the K-(ivDde)-modified SEQ IDNO:518 (50 mg, 0.017 mmol) and diisopropylethylamine (DIEA, 10 mg, 0.08mmol) and the resultant mixture was stirred for 18 hours. Hydrazine (10μL) was added and the solution was stirred for 30 min. The reactionmixture was diluted with water (20 mL), loaded onto a reversed-phase(C18) HPLC column, and eluted with water (0.1% TFA)-acetonitrile (0.1%TFA) system. Fractions containing the required product (>95% purity)were collected and freeze-dried to provide SEQ ID NO:518-(6-PnAO-Glut))(see Scheme 5 as shown in FIG. 11) as a colorless fluffy solid. Theyield was 25.1 mg (47.4%).

2) Preparation of Dimer Containing SEQ ID NO:514 Linked to SEQ ID NO:515

To a solution of the peptide containing SEQ ID NO:515 (a type Bcompound) (10 mg, 0.0034 mmol) and diisopropylethylamine (10 mg, 0.08mmol) in dry DMF (0.2 mL) was added disuccinimidyl glutarate (10 mg,0.031 mmol) and stirred at room temperature for 30 min. The reactionmixture was diluted with ether (3 mL) and stirred. The supernatant wasdecanted, leaving behind the semi-solid in the flask. This process ofwashing the reaction product was repeated with ether (3×5 mL). Thesemi-solid thus obtained was dissolved in dry DMF (0.2 mL) and thepeptide SEQ ID NO:514-(6-PnAO-Glut)) (10 mg, 0.0032 mmol) anddiisopropylethylamine (10 mg, 0.08 mmol) were added and stirred thereaction mixture for 24 h at room temperature. The reaction mixture wasdiluted with water (10 mL), loaded onto a reversed-phase (C18) HPLCcolumn, and eluted with water (0.1% TFA)-acetonitrile (0.1% TFA) system.Fractions containing the required product (>95% purity) were collectedand freeze-dried to provide the heterodimer having SEQ ID NO:514 linkedto SEQ ID NO:515 via a 6-PnAO-Glut linkage (see Scheme 6 as shown inFIG. 12) as a colorless fluffy solid. Yield: 6.7 mg (33%). Thestructures for this and other heterodimers are shown in FIGS. 13A-13C.

Example 9 Cellular Proliferation Assay

Cellular proliferation assays were performed to identify cMet-bindingpeptides that antagonize HGF-stimulated proliferation. These in vitrostudies utilized a leomyosarcoma cell line, SK-LMS-1, in which cellsproliferate in response to HGF. SK-LMS-1 cells were seeded on 96-wellplates at a density of 2000 cells/well. After a 24 hour incubation at 37C, the cells were starved in culture media containing 0.1% BSA insteadof 10% fetal bovine serum for 36 hours at 37 C. Fresh starvation mediawith or without a cMet-binding peptide (10 μM) was added to therespective wells and the cells were incubated for 2 hours at 37 C. DMFwas used as the control vehicle and did not receive a cMet-bindingpeptide. HGF was then added at a concentration of either 50 ng/mL or 100ng/mL and the cells were incubated for an additional 12 hours at 37 C.Proliferation was assessed by measuring the incorporation of BrdU(Calbiochem, San Diego, Calif.) as described by the manufacturer.Results are shown for SEQ ID NO:365 (FIG. 10).

Example 10 Design of a Second Generation cMet-Binding Peptide Library

Initial selection from linear and cyclic peptide libraries identified anumber of positive hits for cMet. The TN9 hits contained a highlyconserved motif (CxGpPxFxC, SEQ ID NO:512, the ‘p’ is less stronglyselected than are the uppercase amino acids). A library was constructedhaving both cyclic and linear members and was built in phage having agene III stump display.

TABLE 1 TN9 and linear components in the second generation library:Libraries of TN9s for cMet (cMet TN9 2nd lib #1) Component 1: TN9consensus with 3 AA left extension                   S   M   G   S   E   T   R   P   T ctcagcagtcactgtcttCC ATG Ggt tct gaa act cgc cct aca                    NcoI..... e   a   g   s   w   h   C   s   G   P   P   t   F   e   C   w   w   yjej jqz jjz ejz zjj qez tgt ejz ggt cct cct eqj ttc jej tgc zjj zjj zez G   T   E   P   T   E   A   S gga acg gag ccg act gaa GCT AGC Gtgactctgacagtctctgt                          NheI... (SEQ ID NO: 518) cMetTN9 2nd lib #2: TN9 consensus with 3 AA right extension.                   S   M   G   S   E   T   R   P   T ctcagcagtcactgtcttcc atg ggt tct gAa act cgc cct AcA                    NcoI..... E   A   G   s   w   h   C   s   G   P   P   t   F   e   C   w   w   yGAG GCT GGT ejz zjj qez tgt ejz ggt cct cct eqj ttc jej tgc zjj zjj zez g   t   e   P   T   E   R   P   S   S   S jjz eqj jej ccg AcT gAA cgtcct agt GCT AGC Gtga ctctgacagtctctgt                                    NheI... (SEQ ID NO: 519) cMet TN92nd lib #3 SIQCKGPPWFSCAMY (SEQ ID NO: 537) with 3 AA extension on left                   S   M   G   S   E   T   R   P   T ctcagcagtcactgtcttcc atg ggt tct gaa act cgc cct AcA                    NcoI..... e   a   g   s   i   q   C   k   G   P   P   w   F   s   C   a   m   yjej jqz jjz ejz ezz qej tgc eej ggt cct cct zjj ttc ejz tgt jqj ezj zez G   T   E   P   T   E   A   S   A ggA Acg gAg ccg AcT gAA GCT AGC Gtgactctgacagtctctgt                         NheI... (SEQ ID NO: 520) cMetTN9 2nd lib #4 SIQCKGPPWFSCAMY (SEQ ID NO: 537) with 3 AA extension onright                    S   M   G   S   E   T   R   P   Tctcagcagtcactgtct tcc atg ggt tct gaa act cgc cct AcA                   NcoI..... E   A   G   s   i   q   C   k   G   P   P   w   F   s   C   a   m   ygag gcc ggt ejz ezz qej tgc eej ggt cct cct zjj ttc ejz tgt jqj ezj zez g   t   e   P   T   E   R   P   S   S   A jjz eqj jej ccg AcT gAA cgtcct agt GCT AGC Gtga ctctgacagtctctgt                                NheI... (SEQ ID NO: 521) cMet TN9 5thlib 330-F05 YYGCKGPPTFECQWM (SEQ ID NO: 531)with 3 AA extension on rightthree peptides have the core sequence CKGPPTFEC (SEQ ID NO: 653)                   S   M   G   S   E   T   R   P   T ctcagcagtcactgtcttcc atg ggt tct gAa act cgc cct AcA                    NcoI..... E   A   G   y   y   g   C   k   G   P   P   t   F   e   C   q   w   mGAG GCT GGT zez zez jjz tgc eej ggt cct cct eqz ttc jej tgt qee zjj ezj g   t   e   P   T   E   R   P   S   S   S jjz eqj jej ccg AcT gAA cgtcct agt GCT AGC Gtga ctctgacagtctctgt                                    NheI... (SEQ ID NO: 522) cMet TN96th lib: 550-G12 AFFCSGPPTFMCSLY (SEQ ID NO: 536) with 3 AA extension onright two peptides have the core sequence CSGPPTFMC (SEQ ID NO: 654)                   S   M   G   S   E   T   R   P   T ctcagcagtcactgtcttcc atg ggt tct gAa act cgc cct AcA                    NcoI..... E   A   G   a   f   f   C   s   G   p   P   t   F   m   C   s   l   yGAG GCT GGT jqz zzq zzq tgt zqz ggt qqj cct eqz ttc ezj tgc ejq qzz zez g   t   e   P   T   E   R   P   S   S   S jjz eqj jej ccg AcT gAA cgtcct agt GCT AGC Gtga ctctgacagtctctgt                                    NheI... (SEQ ID NO: 523) cMet TN97th lib, three AA to left and let first P of gPP vary.                   S   M   G   S   E   T   R   P   T ctcagcagtcactgtcttCC ATG Ggt tct gaa act cgc cct aca                    NcoI..... e   a   g   q   f   k   C   a   G   p   P   s   F   a   C   w   m   tjej jqz jjz qej zzq eej tgt jqz ggt qqj ccg ejz ttc jqq tgt zjj ezj eqq G   T   E   P   T   E   A   S gga acg gag ccg act gaa GCT AGC Gtgactctgacagtctctgt                         NheI... (SEQ ID NO: 524) E= 0.64A + 0.12C + 0.12G + 0.12T Q = 0.12A + 0.64C + 0.12G + 0.12T J= 0.12A + 0.12C + 0.64G + 0.12T Z = 0.12A + 0.12C + 0.12G + 0.64T Note:(0.64)³⁶ = 1.E−7 (0.64)³⁹ = 2.5E−8

Example 11 Analysis of 94-E08 and Other Linear Peptides Selected forBinding cMet

The linear isolate 94-E08 (SEQ ID NO:454) has high affinity for cMet yetthere were few other peptides isolated that had any homology to 94-E08and those that did have very limited similarity over very short regions.Thus, three variable oligonucleotides based on 94-E08 were made: (1)vary the first 13 codons, keeping the last 7 constant; (2) vary 13 ofthe first 18, keeping 5 that showed some similarity to other isolatesfixed; and (3) vary the last 13 codons, keeping the first 5 fixed, seetable 4 below.

TABLE 4 Component #8 with variation in the first 13 positions (SEQ IDNO: 594).               S   M   G   S   E 5′-tcactgtct tCC ATG Ggt tctgaa-    Scab.......| NcoI |  y   d   t   w   v   f   q   f   i   h zezjez eqz zjj jzj zzz qej zzz ezz qez- e   v   p   G   E   L   V   A   M   Q jej jzj qqj ggt gag ctg gtt gctatg cag-  G   G   S   G   T   E   A   S ggt ggt agt ggt act gaa GCT AGCGtga ctctgac-3′                        | NheI  |Scab........ Component#9Fix five AAs and extend variegation to position 18 (SEQ ID NO: 595).              S   M   G   S   E 5′-tcactgtct tCC ATG Ggt tct gaa-   Scab.......| NcoI |  y   D   T   w   v   F   q   f   i   h zez gatact zjj jzj ttt qej zzz ezz qez-  E   V   p   g   e   l   v   a   M   Qgag gtt qqj jjz jej qzj jzj jqj atg caa!  G   G   S   G   T   E   A   Sggt ggt agt ggt act gaa GCT AGC Gtga ctctgac-3′                       | NheI  |Scab........ Component#10 Fix firstseven AAs and vary last 13 (SEQ ID NO: 596).              S   M   G   S   E 5′-tcactgtct tCC ATG Ggt tct gaa-   Scab.......| NcoI |  Y   D   T   W   V   F   Q   F   i   h tat gatact tgg gtt ttt caa ttt ezz qez-  e   v   p   g   e   l   v   a   m   qjej jzz qqj jjz jej qzj jzj jqj ezj qzz!  G   G   S   G   T   E   A   Sggt ggt agt ggt act gaa GCT AGC Gtga ctctgac-3′                       | NheI  |Scab........Oligonucleotide design for construction of the second generation peptidelibrary (SEQ ID NOS:597-646; N.B. oligonucleotides marked “[RC]” consistof the reverse complement of the sequence shown):

vg#1 NcoI.... (CM2_ZTPSAlt) 5′-tcactgtct tcc atg ggt tct gAa- 3′(CM2_TPLalt) 5′-tcactgtct tcc atg ggt tct gAa act cgc cct AcA-3′(CM2_ZTPS) 5′-ctcagcagtcactgtct tcc at-3′ (CM2_TPLong)5′-ctcagcagtcactgtct tcc atg ggt tct gAa act cgc cct AcA-3′ (CM2_V1)5′-tct gAa act cgc cct AcA- jej jqz jjz ejz zjj qez tgt ejz ggt cct ccteqj ttc jej tgc zjj zjj zez- gga acg gag ccg act gaa gct-3′ (CM2_BPL1)[RC] 5′-gga acg gag ccg act gaa GCT AGC Gtga ctctgacagtctctgt-3′(CM2_XBPS) [RC] 5′-CA Gtga ctctgacagtctctgt-3′ (BPL1_CM2) [RC] 5′-ggaacg gag ccg act gaa GCT AGC Gtga ctctgac-3′ (XBPS_CM2) [RC] 5′-act gaaGCT AGC Gtga ctctgac-3′ vg#2 NheI... (CM2_ZTPS) 5′-ctcagcagtcactgtct tccat-3′ (CM2_TPLong) 5′-ctcagcagtcactgtct tcc atg ggt tct gAa act cgc cctAcA-3′ (CM2_V2) 5′-tct gAa act cgc cct AcA- GAG GCT GGT ejz zjj qez tgtejz ggt cct cct eqj ttc jej tgc zjj zjj zez- jjz eqj jej ccg AcT gAA cgtcct agt g-3′ (CM2_2BPL) [RC] 5′-ccg AcT gAA cgt cct agt GCT AGC Gtgactctgacagtctctgt-3′ (CM2_XBPS) [RC] 5′-CA Gtga ctctgacagtctctgt-3′(BPL2_CM2) [RC] 5′-ccg AcT gAA cgt cct agt GCT AGC Gtga ctctgac-3′(XPL2_CM2) [RC] 5′-ct agt GCT AGC Gtga ctctgac-3′ vg#3 (CM2_ZTPS)5′-ctcagcagtcactgtct tcc at-3′ (CM2_TPLong) 5′-ctcagcagtcactgtct tcc atgggt tct gAa act cgc cct AcA-3′ (CM2_V3) 5′-tct gaa act cgc cct AcA- jejjqz jjz ejz ezz qej tgc eej ggt cct cct zjj ttc ejz tgt jqj ezj zez- ggAAcg gAg ccg AcT gAA GC-3′ (CM2_BPL1) [RC] 5′-gga acg gag ccg act gaa GCTAGC Gtga ctctgacagtctctgt-3′ (CM2_XBPS) [RC] 5′-CA Gtgactctgacagtctctgt-3′ vg#4 (CM2_ZTPS) 5′-ctcagcagtcactgtct tcc at-3′(CM2_TPLong) 5′-ctcagcagtcactgtct tcc atg ggt tct gAa act cgc cct AcA-3′(CM2_V4) 5′-tct gaa act cgc cct AcA- gag gcc ggt ejz ezz qej tgc eej ggtcct cct zjj ttc ejz tgt jqj ezj zez- jjz eqj jej ccg AcT gAA cgt cct agtGC-3′ (CM2_2BPL) [RC] 5′-ccg AcT gAA cgt cct agt GCT AGC Gtgactctgacagtctctgt-3′ (CM2_XBPS) [RC] 5′-CA Gtga ctctgacagtctctgt-3′ vg#5(CM2_ZTPS) 5′-ctcagcagtcactgtct tcc at-3′ (CM2_TPLong)5′-ctcagcagtcactgtct tcc atg ggt tct gAa act cgc cct AcA-3′ (CM2_V5)5′-tct gAa act cgc cct AcA-′ GAG GCT GGT zez zez jjz tgc eej ggt cct ccteqz ttc jej tgt qee zjj ezj- jjz eqj jej ccg AcT gAA cgt cct agt GC-3′(CM2_2BPL) [RC] 5′-ccg AcT gAA cgt cct agt GCT AGC Gtgactctgacagtctctgt-3′ (CM2_XBPS) [RC] 5′-CA Gtga ctctgacagtctctgt-3′ vg#6(CM2_ZTPS) 5′-ctcagcagtcactgtct tcc at-3′ (CM2_TPLong)5′-ctcagcagtcactgtct tcc atg ggt tct gAa act cgc cct AcA-3′ (CM2_V6)5′-tct gAa act cgc cct AcA- GAG GCT GGT jqz zzq zzq tgt zqz ggt qqj ccteqz ttc ezj tgc ejq qzz zez jjz eqj jej ccg AcT gAA cgt cct agt GC-3′(CM2_2BPL) [RC] 5′-ccg AcT gAA cgt cct agt GCT AGC Gtgactctgacagtctctgt-3′ (CM2_XBPS) [RC] 5′-CA Gtga ctctgacagtctctgt-3′ vg#7(CM2_ZTPS) 5′-ctcagcagtcactgtct tcc at-3′ (CM2_TPLong)5′-ctcagcagtcactgtct tcc atg ggt tct gAa act cgc cct AcA-3′ (CM2_V7)5′-tct gaa act cgc cct aca- jej jqz jjz qej zzq eej tgt jqz ggt qqj ccgejz zzq jqq tgt zjj ezj eqq- gga acg gag ccg act gaa GC-3′ (CM2_BPL1)[RC] 5′-gga acg gag ccg act gaa GCT AGC Gtga ctctgacagtctctgt-3′(CM2_XBPS) [RC] 5′-CA Gtga ctctgacagtctctgt-3′

Component #8 Vary the First 13 Positions.

(CM2_ZTPSAlt) 5′-tcactgtct tcc atg ggt tct gAa-3′ (CM2C8vg) 5′-tcactgtcttCC ATG Ggt tct gaa - zez jez eqz zjj jzj zzz qej zzz ezz qez - jej jzjqqj ggt gag ctg gtt gct atg cag - ggt ggt agt ggt act gaa GCT(L20botamp) 5′-ggt ggt agt ggt act gaa GCT AGC [RC] Gtga ctct-3′

Component #9 Fix Five AAs and Extend Variegation to Position 18.

(CM2_ZTPSAlt) 5′-tcactgtct tcc atg ggt tct gAa-3′ (CM2C9vg) 5′-tcactgtcttCC ATG Ggt tct gaa - zez gat act zjj jzj ttt qej zzz ezz qez - gag gttqqj jjz jej qzj jzj jqj atg caa - ggt ggt agt ggt act gaa GCT-3′(L20botamp) 5′-ggt ggt agt ggt act gaa GCT AGC [RC] Gtga ctct-3′

Component #10 Fix First Seven AAs and Vary Last 13.

(CM2_ZTPSAlt) 5′-tcactgtct tcc atg ggt tct gAa-3′ (CM2C10vg)5′-tcactgtct tCC ATG Ggt tct gaa - tat gat act tgg gtt ttt caa ttt ezzqez - jej jzz qqj jjz jej qzj jzj jqj ezj qzz - ggt ggt agt ggt act gaaGCT-3′ (L20botamp) 5′-ggt ggt agt ggt act gaa GCT AGC [RC] Gtga ctct-3′

Example 12 Construction of a Second Generation cMet-Binding PeptideLibrary

The phage vector DY3P82 was digested with NheI and NcoI, cleaned andtreated with alkaline phosphatase. The 10 templates, CM2-V1 throughCM2-V7, plus CM2-V8vg, CM2-V9vg and CM2-V10vg, were amplifiedseparately, using the primer pairs listed in Table 5 below.

TABLE 5 Template Sense Antisense CM2_V1 CM2_TPLONG CM2_BPL1 CM2_V2CM2_TPLONG CM2_BPL1 CM2_V3 CM2_TPLONG CM2_BPL1 CM2_V4 CM2_TPLONGCM2_BPL1 CM2_V5 CM2_TPLONG CM2_BPL1 CM2_V6 CM2_TPLONG CM2_BPL1 CM2_V7CM2_TPLONG CM2_BPL1 CM2_V8vg CM2_ZTPSALT L20BOTAMP CM2_V9vg CM2_ZTPSALTL20BOTAMP CM2_V10vg CM2_ZTPSALT L20BOTAMP

Each sample was digested separately with NheI and NcoI, extracted withphenol/chloroform, and mixed in an equimolar ratio prior to performingthe ligation. A vector:insert ratio of 1:5 was used. Ligated DNAconstructs were electroporated into DH5α cells. The resulting librarysize was 1.12×10⁸ different transformants.

Example 13 Measurement of Binding of Peptide Dimers to cMet

Using a BIAcore machine, the binding constants were determined for thepeptide dimers (shown in FIGS. 13A-13C) binding to immobilized cMet-Fc.

Three densities of cMet-Fc (R&D Systems) were cross-linked to thedextran surface of a CM5 sensor chip by the standard amine couplingprocedure (3 μM solution diluted 1:100, 1:50, or 1:20 with 50 mMacetate, pH 5.5). Flow cell 1 was activated and then blocked to serve asa reference subtraction.

Final immobilization levels achieved:R_(L) Fc 2 cMet-Fc=2582R_(L) Fc 3 cMet-Fc=5048R_(L) Fe 4 cMet-Fc=9721

Experiments were performed in PBST buffer (5.5 mM phosphate, pH 7.65,0.15 M NaCl)+0.05% (v/v) Tween-20). Peptide dimers were dissolved indeionized H₂O to 1 mg/mL solutions. Dimers were diluted to 50 nM in PBS.Serial dilutions were performed to produce 25, 12.5, 6.25, and 3.125 nMsolutions. All samples were injected in duplicate. For association,dimers were injected at 30 μL/minute for 3 minutes using the kinjectprogram. Following a 10-minute dissociation, any remaining peptide wasstripped from the cMet surface with two quick injects of 4M MgCl₂ for 2minutes at 50 μL/minute. Sensorgrams were analyzed using BIAevaluationsoftware 3.1. The heterodimer, Ac-GSPEMCMMFPFLYPCNHHAPGGGK{PnAO6-Glut-K[Ac-GSFFPCWRIDRFGYCHANAPGGGKJJ-Glut]-NH₂}-NH₂ (SEQ ID NO514 linked to SEQ ID NO:515), exhibits a K_(D) of 0.79 nM.

Example 14 Enhancing the Serum Residence of cMet-Binding Peptides:Conjugation to Maleimide

It is known in the art that compounds that contain maleimide and othergroups that can react with thiols react with thiols on serum proteins,especially serum albumin, when the compounds are injected. The adductshave serum life times similar to serum albumin, more than 14 days inhumans for example.

Methods are available that allow for the direct synthesis ofmaleimide-labeled linear peptides encompassed by the present invention(Holmes, D. et al., 2000. Bioconjug. Chem., 11:439-444.).

Peptides that include disulfides can be derivatized with maleimide inone of several ways. For example, a third cysteine can be added at thecarboxy terminus. The added cysteine is protected with protecting groupthat is orthogonal to the type of groups used for the cysteines that areto form the disulfide. The disulfide is formed by selectivelydeprotecting the intended cysteines and oxidizing the peptide. The finalcysteine is then deprotected and the peptide reacted with a large molarexcess of a bismaleimide. The resulting compound has one of themaleimides free to react with serum albumin or other thiol-containingserum proteins.

Alternatively, a cyclic peptide of the present invention is synthesizedwith a lysine-containing C-terminal extention, such as -GGGK (SEQ IDNO:513). Lysines of the cMet-binding motif are protected with ivDde andthe C-terminal lysine is deprotected. This lysine is reacted with amaleimide-containing compound, such asN-[e-maleimidocaproyloxy]succinimide ester (Pierce Biotechnology,Rockford, Ill.) or N-[a-Maleimidoacetoxy]succinimide ester (PierceBiotechnology).

Example 15 Enhancing the Serum Residence of cMet-Binding Peptides:Conjugation to a Moiety that Binds Serum Albumin Non-Covalently

Polypeptides having a molecular weight less than 50-60 kDa are rapidlyexcreted. Many small molecules, such as fatty acids, bind to serumalbumin. Attaching a fatty acid or other serum albumin binding moiety toa peptide causes it to bind non covalently to serum albumin and cangreatly prolong serum residence. Fatty acids attached to peptides of thepresent invention should contain at least 12 carbons, preferably atleast 14 carbons and, more preferably at least 16 carbons. The fattyacid could be straight-chain or branched. The fatty acid could besaturated or unsaturated. Palmate (CH₃—(CH₂)₁₄—CO— is a preferred fattyacid. This binding in serum can reduce the rate of excretion (Knudsen,L. et al., 2000. J. Med. Chem., 43:1664-1669). Using methods known inthe art, serum-albumin-binding moieties can be conjugated to any one ofthe peptides or multimeric polypeptide binding constructs hereindisclosed. The serum-albumin-binding moiety can be joined to thecMet-binding peptide through a linker. The linker can be peptidic orotherwise, such as PEG. Linkers of zero to about thirty atoms arepreferred. It is preferred that the linker be hydrophilic. Theserum-albumin-binding moiety can be conjugated to the cMet-bindingpeptide or construct at either end or though a side group of an appendedamino acid. Suitable side groups include lysine and cysteine. Suchcompounds also can comprise, for example, chelators for radionuclides,or other detectable labels or therapeutic constructs, as discussedherein. A cMet peptide or construct joined to a serum-albumin-bindingmoiety will bind cMet.

Example 16 Enhancing the Serum Residence of cMet-Binding Peptides:Conjugation to PEG

Attachment of PEG to proteins and peptides enhances the serum residenceof these molecules. Attachment of PEG (linear or branched) to acMet-binding peptide or multimeric polypeptide construct is expectedgive substantial enhancement of serum residence time. The molecularweight of the PEG be at least 10 kDa, more preferably at least 20 kDa,and most preferably 30 kDa or more. The PEG can be attached at the N- orC-terminus. Methods of attaching PEG to peptides are well known in theart. PEG can be attached to reactive side groups such as lysine orcysteine.

Example 17 Enhancing the Serum Residence of cMet-Binding Peptides:Fusion to Serum Protein

Proteins comprising serum albumin (SA) and other proteins have enhancedserum residence times. The amino-acid sequence of human SA (hSA) isshown in Table 10. Table 11 shows a fusion protein comprising of (SEQ IDNO:657), mature hSA, and SEQ ID NO:658. The cMet-binding peptides areseparated from mature hSA by linkers that are rich in glycine to allowflexible spacing. One need not use all of hSA to obtain an injectableprotein that will have an enhanced serum residence time. Chemicalgroups, such as maleimide and alpha bromo carboxylates, react with theunpaired cysteine (residue 34) to form stable adducts. Thus, one canattach a single chelator to hSA fusion proteins so that the adduct willbind a radionuclide. One can prepare a chelator with a maleimide groupand couple that to hSA or an hSA derivative. Alternatively, hSA or anhSA derivative can be reacted with a bismaleimide and a chelatorcarrying a reactive thiol could be reacted with thebismaleimide-derivatized hSA.

Construction of genes that encode a given amino-acid sequence are knownin the art. Expression of HSA fusions in Saccharomyces cerevisiae isknown in the art.

Example 18 Pretargeting Radioactivity or Toxins to cMet ExpressingTumors

Conventional radioimmune cancer therapy is plagued by two problems. Thegenerally attainable targeting ratio (ratio of administered doselocalizing to tumor versus administered dose circulating in blood orratio of administered dose localizing to tumor versus administered dosemigrating to bone marrow) is low. Also, the absolute dose of radiationor therapeutic agent delivered to the tumor is insufficient in manycases to elicit a significant tumor response. Improvement in targetingratio or absolute dose to tumor would be of great importance for cancertherapy.

The present invention provides methods of increasing active agentlocalization at a target cell site of a mammalian recipient. The methodsinclude, for example, a) administering to a recipient a fusion proteincomprising a targeting moiety and a member of a ligand-anti-ligandbinding pair; b) thereafter administering to the recipient a clearingagent capable of directing the clearance of circulating fusion proteinvia hepatocyte receptors of the recipient, wherein the clearing agentincorporates a member of the ligand-anti-ligand binding pair; and c)subsequently administering to the recipient an active agent comprising aligand/anti-ligand binding pair member.

It is known in the art that hexoses, particularly the hexoses galactose,glucose, mannose, mannose-6-phosphate, N-acetylglucosamine,pentamannosyl phosphate, N-acetylgalactosamine, thioglycosides ofgalactose, and mixtures thereof are effective in causing hepaticclearance. Binding of sugars to hepatic receptors is not, however, theonly means of directing a molecule to the liver.

Clearance of carcinoembryonic antigen (CEA) from the circulation is bybinding to Kupffer cells in the liver. We have shown that CEA binding toKupffer cells occurs via a peptide sequence YPELPK representing aminoacids 107-112 of the CEA sequence. This peptide sequence is located inthe region between the N-terminal and the first immunoglobulin like loopdomain. Using native CEA and peptides containing this sequence complexedwith a heterobifunctional crosslinking agent and ligand blotting withbiotinylated CEA and NCA we have shown binding to an 80 kD protein onthe Kupffer cell surface. This binding protein may be important in thedevelopment of hepatic metastases. (Thomas, P. et al., 1992. Biochem.Biophys. Res. Commun., 188:671-677)

To use YPELPK (SEQ ID NO:655) as a clearance agent, one fuses thissequence via a linker to a moiety that binds the fusion protein (Ab).For example, if the Ab has affinity for DOTA/Re, one would make aderivative having YPELPK attached to DOTA/Re; for example,rvYPELPKpsGGG-DOTA. ‘rvYPELPKps’ is a fragment of CEA which includes theYPELPK sequence identified by Thomas et al. (supra). Any convenientpoint on DOTA can be use for attachment. RVYPELPKPSGGG-DOTA/cold Re (SEQID NO:656) would then be used as a clearing agent. The Fab correspondingto the fusion Ab would have affinity for the clearing agent of Kd<100nM, preferably Kd<10 nM, and most preferably Kd<1 nM.

The therapeutic agent would contain DOTA/¹⁸⁵Re. In a preferredembodiment, the therapeutic agent would contain two or more DOTAmoieties so that the Ab immobilized on the tumor would bind the bis-DOTAcompound with high avidity. The two DOTA moieties would preferably beconnected with a hydrophilic linker of ten to thirty units of PEG. PEGis a preferred linker because it is not degraded, promotes solubility.Ten to thirty units of PEG is not sufficient to give the bis DOTAcompound a very long serum residence time. A half life of 30 minutes to10 hours is acceptable. The serum half life should be longer than theradioactive half life of the radionuclide used so that most of theradiation is delivered to the tumor or to the external environment.

In one embodiment, a “fusion protein” of the present invention comprisesat least one Wet-binding peptide fused to the amino terminus or thecarboxy terminus of either the light chain (LC) or the heavy chain (HC)of a human antibody. Optionally and preferably, two or more cMet-bindingpeptides are fused to the antibody. The antibody is picked to have highaffinity for a small molecule that can be made radioactive or have atoxin attached. Preferably, the affinity of the Fab corresponding to theAb has affinity for the small molecule with K_(d) less than 100 nM, morepreferably less than 10 nM, and most preferably less than 1 nM. Thesmall molecule could be a chelator capable of binding a usefulradioactive atom, many of which are listed herein. The small moleculecould be a peptide having one or more tyrosines to which radioactiveiodine can be attached without greatly affecting the binding property ofthe peptide.

Any cMet-binding peptide (CMBP) of the present invention can be fused toeither end of either chain of an antibody that is capable of binding asmall radioactive compound. Useful embodiments include:

1) CMBP#1::link::LC/HC,2) LC::link::CMBP#1/HC,3) LC/CMBP#1::link::HC,4) LC/HC::link::CMBP#1,5) CMEBP#1::link1::LC::link2::CMBP#2/HC,6) LC/CMBP#1::link1::HC::link2::CMBP#2,7) CMBP#1::link1::LC/CMBP#2::link2::HC,8) CMBP#1::link1::LC/HC::link2:: CMBP#2,9) LC::link1::CMBP#1/CMBP#2::link2::HC,10) LC::link1::CMBP#1/HC::link2:: CMBP#2,11) CMBP#1::link1::LC::link2::CMBP#2/CMBP#3::link3::HC,12) CMBP#1::link1::LC::link2::CMBP#2/HC::link3::CMBP#3,13) CMBP#3::link3::LC/CMBP#1::link114) LC::link3::CMBP#3/CMBP#1::link1::HC::link2::CMBP#2, and15) CMBP#1::link1::LC::link2::CMBP#2/CMBP#3::link3::HC: link4:: CMBP#4.In cases (5)-(15), the linkers (shown as “link1”, “link2”, “link3”, and“link4”) can be the same or different or be absent. These linkers, ifpresent, are preferably hydrophilic, protease resistant, non-toxic,non-immunogenic, and flexible. Preferably, the linkers do not containglycosylation sites or sequences known to cause hepatic clearance. Alength of zero to fifteen amino acids is preferred. The cMet-bindingpeptides (CMBP#1, #2, #3, and #4) could be the same or different. If theencoded amino-acid sequences are the same, it is preferred that the DNAencoding these sequences is different.

Since antibodies are dimeric, each fusion protein will present twocopies of each of the fused peptides. In case (15), there will be eightCMBPs present and binding to cMet-displaying cells should be highlyavid. It is possible that tumor penetration will be aided by moderatecMet affinity in each of the CMBPs rather than maximal affinity.

The fusion protein is produced in eukaryotic cells so that the constantparts of the HC will be glycosylated. Preferably, the cells aremammalian cells, such as CHO cells.

The fusion proteins are injected into a patient and time is allowed forthe fusion protein to accumulate at the tumor. A clearing agent isinjected so that fusion protein that has not become immobilized at thetumor will be cleared. In previous pretargeting methods, the antibodycombining site has been used to target to the tumor and biotin/avidin orbiotin/streptavidin has been used to attach the radioactive or toxicagent to the immobilized antibody. The biotin/avidin or streptavidinbinding is essentially irreversible. Here we fuse a target-bindingpeptide to the antibody which is picked to bind a radioactive or toxicagent. Because the fusion protein contains 2, 4, 6, or 8 CMBPs, bindingof the fusion protein to the tumor is very avid. A clearing agent thatwill cause fusion protein not immobilized at the tumor to clear can beadministered between 2 and 48 hours of the injection of the fusionprotein. Because the clearance agent is monomeric in the moiety thatbinds the antibody, complexes of clearance agent and immobilized fusionprotein will not have very long life times. Within 4 to 48 hours ofinjecting clearance agent, the immobilized antibody will have lost anyclearance agent that binds there. The active agent is, preferably,dimeric in the moiety that binds the fusion protein. The active agent isinjected between 2 and ˜48 hours of injection of clearance agent.

Example 19 Binding of cMet Binding Peptides/Avidin HRP Complex toMDA-MB-231 Cells

The spacer length requirements for the binding of a biotinylatedderivative of a cMet binding peptide, SEQ ID NO:514, to cMet expressingMDA-MB-231 cells were determined. In order to decide the spacer lengthto be placed in between peptide and biotin, derivatives were synthesizedwith no spacer, a single spacer, J, and two spacers, JJ. These threedifferent derivatives of cMet-binding peptide SEQ ID NO:514 and acontrol peptide that does not bind to cMet, were tested as tetramericcomplexes with neutravidin HRP for their ability to bind cMet expressingMB-231 cells. All three tetrameric complexes of cMet-binding peptidesbound to the MB231 cells as compared to control peptide; however, thepeptide with the JJ spacer exhibited the best K_(D) (12.62 nM). Thissuggests that inclusion of two spacers (JJ) between the cMet-bindingpeptide and the biotin is better than one or no spacer.

Cell Culture: MDA-MB231 cells were obtained from ATCC and grown asmonolayer culture in their recommended media plus 1 mL/L pen/strep(InVitrogen, Carlsbad, Calif.). Cells were split the day before theassay, 35000 cells were added to each well of a 96-well plate.

Binding of Peptide/Neutravidin HRP to MDA-MB-231 Cells

Complexes of control peptide, and the SEQ ID NO:514 derivativesdescribed above, with neutravidin-HRP, were prepared as described aboveand tested for their ability to bind MDA-MB-231 cells. During thepeptide/neutravidin-HRP complex preparation, a 7.5-fold excess ofbiotinylated peptides over neutravidin-HRP was used to make sure thatall four biotin binding sites on neutravidin were occupied. Aftercomplex formation, the excess of free biotinylated peptides was removedusing soft release avidin-sepharose to avoid any competition betweenfree biotinylated peptides and neutravidin HRP-complexed biotinylatedpeptides. The experiment was performed at several differentconcentrations of peptide/neutravidin-HRP, from 0.28 nM to 33.33 nM, togenerate saturation binding curves for derivatives without a J spacerand with a single J spacer (FIG. 14), and 0.28 nM to 16.65 nM togenerate a saturation binding curve for the derivative with the JJspacer (FIG. 14). In order to draw the saturation binding curve, thebackground binding of the control peptide/neutravidin HRP complex wassubtracted from the binding of the SEQ ID NO:514 derivatives in complexwith neutravidin-HRP for each concentration tested. Therefore,absorbance on the Y-axis of FIG. 14 is differential absorbance(cMet-binding peptide minus control peptide) and not the absoluteabsorbance. Analysis of the saturation binding data in FIG. using GraphPad Prism software (version 3.0) yielded a K_(D) of 12.62 nM (+/−3.16)for the tetrameric derivative with the JJ spacer, 155.4 nM (+/−86.56)for the tetrameric derivative with the J spacer and 123.8 nM (+/−37.71)for the tetrameric derivative without a spacer peptide complexes. Thesebinding constants are, as expected, lower than that measured by FP forthe related monodentate peptide SEQ ID NO:514 (880 nM).

Results: It is evident from FIG. 14 that the derivative with the JJspacer showed much better binding to cMet on MDA-MB-231 cells thaneither of the other two derivatives, with a K_(D) of 12.62 nM aftersubtracting binding of control peptide as background binding (n=1). Thissuggests that a certain minimum spacer length may be required to be ableto reach multiple different binding sites on cells and thus achievemultimeric binding. This minimum spacer length could depend on thespacing between different target molecules on cells. As was the casewhere the binding target was KDR, the neutravidin-HRP assay withbiotinylated peptides identified with phage display was useful foridentifying peptides capable of binding to an immobilized target evenwhen the affinity of the monomeric binding sequence is too low for anELISA-type assay (with washing steps after binding) to work well.

TABLE 6 cMet-binding peptide sequences SEQ ID NO: Isolate Sequence CLASSI TN6: SEQ ID NO: 001 571-C05, GSWIICWWDNCGSSAP SEQ ID NO: 002 465-A06,GSYYDCREFQCNKPAP SEQ ID NO: 003 465-D09, GSSHLCNPEFCHFTAP SEQ ID NO: 004569-H10, GSMLMCELWWCRFLAP SEQ ID NO: 005 470-E11, GSLIFCPYGECMMYAP SEQID NO: 006 452-F01, GSEYSCRTSRCIFSAP SEQ ID NO: 007 569-C03,GSFILCWWTFCDTNAP SEQ ID NO: 008 574-H03, GSSTICPGTACVDHAP SEQ ID NO: 009567-C08, GSLIICWWSWCDKQAP SEQ ID NO: 010 561-C08, GSFNICPYQWCTLWAPConsensus Motif: G-S-X1-X2-X3-C-X4-X5-X6-X7-C-X8-X9-X10- A-P-G-G-K;where X1 is F, L, S, W, Y, or M; X2 is I, Y, H, T, or N; X3 is I, L, D,M, F, or S, preferably I; X4 is P, R, W, N, or E, preferably W or P; X5is W, Y, E, P, L, T, or G; X6 is S, T, D, F, E, W, G, or Q; X7 is F, W,N, Q, E, R, or A; X8 is G, N, H, R, M, I, D, V, or T; X9 is S, K, F, M,T, D, or L; and X10 is S, P, T, L, Y, N, H, Q, or W. CLASS II TN8: SEQID NO: 011 573-F04, AGGFACGPPWDICWMFGT SEQ ID NO: 012 570-E07,AGAWNCEYPTFICEWQGA SEQ ID NO: 013 456-E04, AGNWICNLSEMRCYPKGT SEQ ID NO:014 434-E12, AGDGWCMAWPEICEWLGT SEQ ID NO: 015 489-A04,AGLYLCDLSIMYCFFQGT SEQ ID NO: 016 484-D08, AGWWSCQWELNVCIWQGT SEQ ID NO:017 482-D02, AGYYHCIDDFPQCKWMGT SEQ ID NO: 018 437-A09,AGWFECEFGFWGCNWLGT SEQ ID NO: 019 352-E04, AGTVYCSWESSECWWVGT SEQ ID NO:020 376-E05, AGVWICRVWDDECFFQGT SEQ ID NO: 021 482-A12,AGDHYCWEEWWFCWDSGT SEQ ID NO: 022 423-C11, AGVLQCIGFEWFCDIWGT SEQ ID NO:023 499-C09, AGVIVCNLSMMYCLYPGT SEQ ID NO: 024 457-A09,AGYPECKDNYHWCEWKGT SEQ ID NO: 025 573-E07, AGWTWCDLSMMSCIFHGT SEQ ID NO:026 465-F08, AGVTNCNLSTMFCFLHGT SEQ ID NO: 027 465-E09,AGTLSCSEEYKSCQLQGT SEQ ID NO: 028 444-B08, AGTIRCNLAMMVCMFEGT SEQ ID NO:029 465-E11, AGQYLCTQAALGCPEWGT SEQ ID NO: 030 465-D12,AGQMWCAEKNSKCYQWGT SEQ ID NO: 031 470-A02, AGQAVCEWGPFWCQMQGT SEQ ID NO:032 465-C01, AGPYSCHSESHDCKLMGT SEQ ID NO: 033 448-H02,AGPLFCFEWPSLCHWGGT SEQ ID NO: 034 465-D01, AGNLPCHWNMSVCDHQGT SEQ ID NO:035 571-C11, AGMDFCEGFWFLCIGNAT SEQ ID NO: 036 465-B11,AGLLGCWDMPMECTGEGT SEQ ID NO: 037 442-E08, AGKYMCEGFEWFCEMWGT SEQ ID NO:038 465-C11, AGKTVCQKWESVCSGMGT SEQ ID NO: 039 465-F10,AGKQWCVVWEETCDQLGT SEQ ID NO: 040 471-A11, AGIWFCNNEEKSCWAYGT SEQ ID NO:041 465-C07, AGHTICQHKALGCPANGT SEQ ID NO: 042 465-D04,AGHFECPKHQYMCDMPGT SEQ ID NO: 043 445-E04, AGGNWCSFYEELCEWLGT SEQ ID NO:044 465-B06, AGGHWCLELKHLCPPYGT SEQ ID NO: 045 470-C02,AGFWDCGWMMQDCHMHGT SEQ ID NO: 046 458-B05, ADAWMCEYFQWNCGDKGT SEQ ID NO:047 545-E08, GDGFLCRWENGWCEFWDP Consensus Motif:A-G-X1-X2-X3-C-X4-X5-X6-X7-X8-X9-C-X10- X11-X12-G-T-G-G-G-K; where X1 isany amino acid other than C, preferably G, V, W, T, K, Q; X2 is anyamino acid other than C, preferably W, Y, L, F, T; X3 is any amino acidother than C, preferably W, E, F, I, L, S X4 is any amino acid otherthan C, preferably E, N, Q; X5 is any amino acid other than C,preferably W, L, E; X6 is any amino acid other than C, preferably E, S,Y; X7 is any amino acid other than C, preferably E, M, P; X8 is anyamino acid other than C, preferably M, S, W; X9 is any amino acid otherthan C, preferably F, L, V; X10 is any amino acid other than C,preferably E, D, W; X11 is any amino acid other than C, preferably W, F,M; and X12 is any amino acid other than C, preferably Q, W, L. CLASS IIITN9 #1: SEQ ID NO: 048 325-H05, AGSIQCKGPPWFSCAMYGT SEQ ID NO: 049330-F05, AGYYGCKGPPTFECQWMGT SEQ ID NO: 050 333-F09, AGQFKCAGPPSFACWMTGTSEQ ID NO: 051 336-G04, AGWFQCKGPPSFECERHGT SEQ ID NO: 052 334-G06,AGWTHCIGPPTFECIPMGT SEQ ID NO: 053 330-B07, AGSFACKGPPTFACVEFGT SEQ IDNO: 054 330-C10, AGNYFCAGSPSFSCYFMGT SEQ ID NO: 055 331-G04,AGSWHCAGPPSFECWEFGT SEQ ID NO: 056 548-F06, AGWISCAGPPTFACWPGGT SEQ IDNO: 057 538-F08, AGFVNCKGPPTFECILTGT SEQ ID NO: 058 547-H07,AGDWICHGPPMFECEWVGT SEQ ID NO: 059 323-A11, AGYTSCVGPPSFECTPYGT SEQ IDNO: 060 333-H03, AGYFECKGPPTFECWLSGT SEQ ID NO: 061 329-D02,AGHAWCSGPPRFECWPPGT SEQ ID NO: 062 550-C09, AGHYWCAGPPTFICMGPGT SEQ IDNO: 063 548-E08, AGETTCLGWPTFVCVDYGT SEQ ID NO: 064 332-A05,AGHGTCRGWPTFECIYFGT SEQ ID NO: 065 330-C01, AGDWHCQGPPAFMCWMIGT SEQ IDNO: 066 545-A09, AGLPKCSGPPWFSCYYGGT SEQ ID NO: 067 334-C08,AGGWECTGPPWFQCGYYGT SEQ ID NO: 068 333-C05, AGDIVCTGHPYFECWSWGT SEQ IDNO: 069 551-B02, AGTWHCAGPPWFTCYMDGT SEQ ID NO: 070 551-G12,AGSWECTGPPSFHCQWYGT SEQ ID NO: 071 330-G09, AGHWICVGPPTFSCQWHGT SEQ IDNO: 072 331-F01, AGEWWCHGPPEFLCYWTGT SEQ ID NO: 073 274-B07,AGETVCYWLNGWFCVDDGT SEQ ID NO: 074 335-D11, AGSIQCVGPPSFECTPYGT SEQ IDNO: 075 336-D07, AGYSVCKGYPSFECAFFGT SEQ ID NO: 076 332-C03,AGVNSCLGPPTFECYQMGT SEQ ID NO: 077 331-D03, AGYWHCKGPPHFACEFHGT SEQ IDNO: 078 331-G06, AGNWICTGPPSFGCWYHGT SEQ ID NO: 079 552-G03,AGYWSCAGPPMFMCTWQGT SEQ ID NO: 080 552-G11, AGYWDCKGPPHFFCEWHGT SEQ IDNO: 081 550-G08, AGYFHCSGSPWFQCDYYGT SEQ ID NO: 082 550-G12,AGWYNCSGENFWNCKWIGT SEQ ID NO: 083 552-A01, AGWSDCLGPPQFTCVHWGT SEQ IDNO: 084 548-C06, AGTMYCLGPPTFICQQYGT SEQ ID NO: 085 545-B12,AGSYWCSGPPTFMCRYEGT SEQ ID NO: 086 549-F06, AGSTDCRGHPTFECWGWGT SEQ IDNO: 087 552-F01, AGSSPCKGWPTFECYFYGT SEQ ID NO: 088 547-H12,AGSIACTGWPYFSCIDLGT SEQ ID NO: 089 550-F11, AGQFYCSGPPTFQCIMIGT SEQ IDNO: 090 548-D08, AGPWKCTGPPTFSCIQFGT SEQ ID NO: 091 549-D02,AGNYWCSGPPSFICHAVGT SEQ ID NO: 092 552-F02, AGMTLCAGPPTFECYEVGT SEQ IDNO: 093 545-E04, AGETKCSGPPYFYCWMEGT SEQ ID NO: 094 545-E05,AGETFCVGNPSFECWSWGT SEQ ID NO: 095 547-H03, AGETFCSGWPTFECMQWGT SEQ IDNO: 096 552-G09, AGEIFCVGPPTFTCMWTGT SEQ ID NO: 097 550-A08,AGDFICQGPPSFVCTNIGT SEQ ID NO: 098 550-G07, AGAFFCSGPPTFMCSLYGT SEQ IDNO: 099 551-A05, AGWGWCSGPPMFMCTEYGT SEQ ID NO: 100 548-C10,GSEFECTGWPEFRCYEYAP SEQ ID NO: 101 465-C10, GSILYCINRNDPQCPYTAPConsensus Motif: G-X1-X2-X3-C-X4-G-X5-P-X6-F-X7-C-X8-X9- X10-G-T; where:X1 is any amino acid other than C, preferably E, S, Y, or W; X2 is anyamino acid other than C, preferably W, T, or F; X3 is any amino acidother than C, preferably W, H, or F; X4 is any amino acid other than C,preferably A, K, S, or T; X5 is any amino acid other than C, preferablyP or W; X6 is any amino acid other than C, preferably T or S; X7 is anyamino acid other than C, preferably E or S; X8 is any amino acid otherthan C, preferably W, Y, or I; X9 is any amino acid other than C,preferably W, Y, M, or E; and X10 is any amino acid other than C,preferably Y. CLASS IV TN9 #2: SEQ ID NO: 102 605-G10,SETRPTEAGDLICSGPPTFICTLYHTEPTE SEQ ID NO: 103 593-C01,SETRPTQAVRSQCSGPPTFECWYFGTEPTE SEQ ID NO: 104 592-C01,SETRPTEGGSWYCSGPPAFECWWYGTEPTE SEQ ID NO: 105 591-E01,SETRPTVASRWHCNGPPTFECWRYGTEPTE SEQ ID NO: 106 590-E01,SETRPTEAGTFHCSGPPTFECWSYGPKPTE SEQ ID NO: 107 589-B01,SETRPTEAGSLWCMGPPWFCCVIYGTQPTE SEQ ID NO: 108 607-A02,SETRPTEAGILHCSGPPTFECWWNYTEPTE SEQ ID NO: 109 590-F01,SETRPTESGRVHCPGPPWFRCARNGTEPTE SEQ ID NO: 110 589-C01,SETRPTAAGRILCTGPPWFSCAMYGTEPTE SEQ ID NO: 111 606-B11,SETRPTEAADWLCSGPPTFECWWFGTEPTE SEQ ID NO: 112 593-E01,SETRPTQVGRWQCDGPPTFACRSYGTEPTE SEQ ID NO: 113 592-F12,SETRPTEAGSTKCSGPPTFECWWFDTEPTE SEQ ID NO: 114 590-F07,SETRPTVAGSWHCSGPPTFECWWYGTEPTE SEQ ID NO: 115 588-D02,SETRPTEAGRNHCKGPPGFRCAMTDTEPTE SEQ ID NO: 116 607-H09,SETRPTETDFVYCRGPPTFECWWYGTEPTE SEQ ID NO: 117 590-H01,SETRPTSSGSRHCKGPPTFECWGYGTEPTE SEQ ID NO: 118 589-F01,SETRPTEAGSWRCSGPPTFECWWYETSPTE SEQ ID NO: 119 608-F11,SETRPTDAIRSYCSGPPTFECWWFGTEPTE SEQ ID NO: 120 606-D11,SETRPTEAGSWNCSGPPAFECWWYGSEPTE SEQ ID NO: 121 604-D04,SETRPTEAGSWQCSGPPTFECWSFGTEPTE SEQ ID NO: 122 602-A11,SETRPTEAGSWHCNGPPTFECWWYDMEPTE SEQ ID NO: 123 593-F02,SETRPTEAGRVSCLGPPTFECWWFVPEPTE SEQ ID NO: 124 591-H05,SETRPTDAGSWRCAGPPTFECWWFGTEPTE SEQ ID NO: 125 590-H06,SETRPTEPVTWQCTGPPTFECWWLGTEPTE SEQ ID NO: 126 588-F10,SETRPTDAVSTHCNGPPTFECYIYGTEPTE SEQ ID NO: 127 608-G03,SETRPTVAESWYCVGPPSFECWWYGTEPTE SEQ ID NO: 128 604-D09,SETRPTEAGSWNCSGPPTFECWSYQTEPTE SEQ ID NO: 129 602-A12,SETRPTEAGSGHCNGPPTFKCWWYDMEPTE SEQ ID NO: 130 592-G11,SETRPTDQDSWQCSGPPTFECWWYGTEPTE SEQ ID NO: 131 588-G01,SETRPTESTQVQCAGPPSFACWMTGTEPTE SEQ ID NO: 132 606-E05,SETRPTEVESWHCSGPPTFECWWYGTEPTE SEQ ID NO: 133 594-C07,SETRPTEAGSFHCSGPPTFECWLYWTDPTE SEQ ID NO: 134 592-H01,SETRPTEAGQFGCKGPPPFECKLMGRVPTE SEQ ID NO: 135 605-C05,SETRPTDTVTWHCNGPPTFECWWYGTEPTE SEQ ID NO: 136 594-E08,SETRPTEADRWHCDGPPTFECWWYGTEPTE SEQ ID NO: 137 593-B11,SETRPTEAGSIQCVGPPWFSCRMYVTEPTE SEQ ID NO: 138 590-C01,SETRPTVSGSWQCVGPPTFECWSYGTEPTE SEQ ID NO: 139 612-G11,SETRPTENGSWHCNGPPTFECWWYGTEPTE SEQ ID NO: 140 612-E08,SETRPTEAGSWHCSGPPIFECWWYDMEPTE SEQ ID NO: 141 612-A02,SETRPTVDGGWHCNGPPTFECWMYGTEPTE SEQ ID NO: 142 611-G01,SETRPTDAGTWNCTGPPSFECWWFGTEPTE SEQ ID NO: 143 610-G04,SETRPTWDGKWHCSGPPTFECWWYGTEPTE SEQ ID NO: 144 610-E06,SETRPTEAGSWRCSGPPTFECWWYYTEPTE SEQ ID NO: 145 610-C06,SETRPTEAGNWLCSGPPTFECWWYVTGPTE SEQ ID NO: 146 610-A04,SETRPTEGGNWHCSGPPTFECWLYGTEPTE SEQ ID NO: 147 612-D02,SETRPTEAGGWHCSGPPTFECWWFNNEPTE SEQ ID NO: 148 612-A12,SETRPTEVISWHCSGPPTFECYRYGTEPTE SEQ ID NO: 149 611-D03,SETRPTEVGSWHCNGPPTFECWWYGTEPTE SEQ ID NO: 150 610-G10,SETRPTLASTWYCSGPPTFECWWYGTEPTE SEQ ID NO: 151 610-A11,SETRPTEAGGWYCKGPPTFECWWDGTEPTE SEQ ID NO: 152 612-H02,SETRPTEAGGWFCSGPPTFECWWYDTVPTE SEQ ID NO: 153 612-B01,SETRPTEAATWQCSGPPTFECWGYGTEPTE SEQ ID NO: 154 610-C12,SETRPTEAGDYVCVGPPTFECYLMDAEPTE SEQ ID NO: 155 610-B01,SETRPTEAGGWYCSGPPSFECWSYGTEPTE SEQ ID NO: 156 612-H04,SETRPTESSSWHCSGPPTFECWRFGTEPTE SEQ ID NO: 157 612-B09,SETRPTEAGSWYCSGPPTFECWWYYAEPTE SEQ ID NO: 158 611-G07,SETRPTLAGNWQCSGPPTFECWWYGTEPTE SEQ ID NO: 159 611-E10,SETRPTEAGSWHCNGPPTFECWQYGTEPTE SEQ ID NO: 160 610-H02,SETRPTEAGSWECHGPPSFECWWYGTEPTE SEQ ID NO: 161 610-D03,SETRPTEAGSWRCSGPPTFECWWYDAEPTE SEQ ID NO: 162 610-B03,SETRPTEAGSWNCAGPPTFECWWYGTEPTE SEQ ID NO: 163 612-H05,SETRPTEAGSFYCSGPPTFECWQYVPEPTE SEQ ID NO: 164 612-F05,SETRPTEAGSWMCSGPPTFECWQYFTEPTE SEQ ID NO: 165 612-B10,SETRPTEAGSLHCSGPPTFECWWWETEPTE SEQ ID NO: 166 611-E11,SETRPTEEGVWHCNGPPTFECWWYGTEPTE SEQ ID NO: 167 610-F08,SETRPTEAGRWNCSGPPTFECWWYSTEPTE SEQ ID NO: 168 610-D05,SETRPTEAGSWRCSGPPTFECWWFGTEPTE SEQ ID NO: 169 610-B04,SETRPTQAVSSYCSGPPTFECWSFGTEPTE SEQ ID NO: 170 612-B12,SETRPTEAGRSYCSGPPTFECWWYATEPTE SEQ ID NO: 171 611-H01,SETRPTVVAKVHCAGPPTFECWTYGTEPTE SEQ ID NO: 172 610-H05,SETRPTEPGSWHCSGPPTFVCWWWGTEPTE SEQ ID NO: 173 610-F10,SETRPTEAGRWHCSGPPTFECWWHDTEPTE SEQ ID NO: 174 612-H07,SETRPTEAGSWQCTGPPTFECWGYVEEPTE SEQ ID NO: 175 612-G09,SETRPTEAGSWQCGGPPTFECWWYYTGPTE SEQ ID NO: 176 612-F08,SETRPTEAGSWYCTGPPTFECWLYETYPTE SEQ ID NO: 177 611-H08,SETRPTAAWSGSCSGPPSFECWNYGTEPTE SEQ ID NO: 178 610-E01,SETRPTEAGSWQCSGPPTFACWWYGTEPTE SEQ ID NO: 179 610-B09,SETRPTEAGILHCSGPPTFECWWEVMEPTE SEQ ID NO: 180 612-E07,SETRPTEAGRVACSGPPTFECWSYDEEPTE SEQ ID NO: 181 612-C11,SETRPTEAGNWECQGPPTFECWWFGTEPTE SEQ ID NO: 182 610-E04,SETRPTLASNGYCNGPPTFECWHYGTEPTE SEQ ID NO: 183 610-B12,SETRPTEAGSFHCSGPPTFECIWYGSEPTE SEQ ID NO: 184 616-B11,SETRPTEAGSWYCSGPPTFACWWDGTEPTE SEQ ID NO: 185 615-H08,SETRPTQGDNWNCSGPPTFECWWYGTEPTE SEQ ID NO: 186 615-B11,SETRPTEAGRWHCNGPPTFECWRYDYDPTE SEQ ID NO: 187 614-C07,SETRPTEAYSWECTGPPMFECWWYGTEPTE SEQ ID NO: 188 613-H12,SETRPTEVVDWHCSGPPQFECWWYGTEPTE SEQ ID NO: 189 613-F02,SETRPTEAGSWNCSGPPTFECWWYGSEPTE SEQ ID NO: 190 613-D05,SETRPTASGSWHCSGPPTFECWIFGTEPTE SEQ ID NO: 191 612-H12,SETRPTEAGAWYCMGPPTFECWWYDRGPTE SEQ ID NO: 192 616-D05,SETRPTEAGGLHCSGPPTFECWWYDTEPTE SEQ ID NO: 193 615-C01,SETRPTVGGSWDCKGPPTFECWSYGTEPTE SEQ ID NO: 194 614-E09,SETRPTEAGAWSCLGPPTFECWWYGTEPTE SEQ ID NO: 195 614-A03,SETRPTEAGSLHCSGPPTFECWWFDTEPTE SEQ ID NO: 196 616-C02,SETRPTAGRSWECSGPPTFECWVFGTEPTE SEQ ID NO: 197 615-C04,SETRPTDNGSWHCNGPPTFECWWYGTEPTE SEQ ID NO: 198 614-C12,SETRPTEAGSWQCKGPPTFECWWYGTEPTE SEQ ID NO: 199 615-C11,SETRPTEVGNYKCSGPPTFECWWYGTEPTE SEQ ID NO: 200 614-H08,SETRPTEAGSWHCVGPPTFECWGYVTEPTE SEQ ID NO: 201 614-E11,SETRPTEAGSFVCKGPPTFECYWFGQDPTE SEQ ID NO: 202 616-E10,SETRPTEAGSWHCSGPPTFECWWYGPDPTE SEQ ID NO: 203 615-D02,SETRPTEAERWHCSGPPTFECWWYGTEPTE SEQ ID NO: 204 614-F04,SETRPTEAGSWHCSGPPTFECWFYVKEPTE SEQ ID NO: 205 614-D06,SETRPTEAGSWDCSGPPTFECWWFGTEPTE SEQ ID NO: 206 614-B08,SETRPTEPAGWECRGPPSFECLWYGTEPTE SEQ ID NO: 207 613-H01,SETRPTDAGPWNCTGPPSFECWWYGTEPTE SEQ ID NO: 208 613-E04,SETRPTEARGWHCSGPPTFECWLWGTEPTE SEQ ID NO: 209 613-B08,SETRPTEAGRWNCSGPPTFECWQYEMDPTE SEQ ID NO: 210 615-D04,SETRPTEAGSWYCSGPPTFECFWYDTEPTE SEQ ID NO: 211 615-A05,SETRPTESGSWHCSGPPTFECWWFGTEPTE SEQ ID NO: 212 614-E04,SETRPTEAGSWLCTGPPTFECWWFDTDPTE SEQ ID NO: 213 613-E06,SETRPTEPSHWHCVGPPTFACWWYVTDPTE SEQ ID NO: 214 613-C05,SETRPTEAGSWYCSGPPMFECYLFVTEPTE SEQ ID NO: 215 616-C07,SETRPTEAVNWHCLGPPSFECWQFGTEPTE SEQ ID NO: 216 615-G02,SETRPTEAGSWHCSGPPTFECWWYGTDPTE SEQ ID NO: 217 615-E06,SETRPTEAGSWHCSGPPTFECWSFVSLPTE SEQ ID NO: 218 615-A08,SETRPTEGSEWSCIGPPSFECWWYGTEPTE SEQ ID NO: 219 614-G01,SETRPTEDGYWNCSGPPTFECWWHGTEPTE SEQ ID NO: 220 613-D01,SETRPTEAGSWSCSGPPTFECWPYYTEPTE SEQ ID NO: 221 614-G02,SETRPTEAGSWYCSGPPTFECWWYWPEPTE SEQ ID NO: 222 614-E06,SETRPTDDGRWSCAGPPTFECWRYGTEPTE SEQ ID NO: 223 620-E11,SETRPTEGGSWSCGGPPTFECWWFGTEPTE SEQ ID NO: 224 620-A11,SETRPTVTGSWYCSGPPTFECWWYGTEPTE SEQ ID NO: 225 618-F04,SETRPTEASSWYCTGPPAFECWWYGTEPTE SEQ ID NO: 226 617-G06,SETRPTEAGSWLCSGPPTFECWWYGTEPTE SEQ ID NO: 227 616-G06,SETRPTESVRWYCSGPPTFECWWYGTEPTE SEQ ID NO: 228 620-F10,SETRPTEAGRLVCSGPPTFMCRTYATDPTE SEQ ID NO: 229 619-G04,SETRPTEAGSWECTGPPWFVCRQYAIEPTE SEQ ID NO: 230 618-F12,SETRPTEAGYLYCSGPPTFECWWYDTMPTE SEQ ID NO: 231 618-B06,SETRPTEAGSWHCSGPPTFECWWFGTEPTE SEQ ID NO: 232 617-E09,SETRPTEAGNWHCLGPPTFECWWYGTEPTE SEQ ID NO: 233 616-F10,SETRPTEAGSWHCSGPPTFECWWYDTEPTE SEQ ID NO: 234 620-B11,SETRPTESGGWYCSGPPAFECWWYGTEPTE SEQ ID NO: 235 619-G07,SETRPTVAGAVSCSGPPTFECWWYGTEPTE SEQ ID NO: 236 619-E11,SETRPTEAGRWYCSGPPTFECWWFLPDPTE SEQ ID NO: 237 619-B12,SETRPTEAGGWHCSGPPSFECWWFDTVPTE SEQ ID NO: 238 618-G11,SETRPTGVGGWYCSGPPSFECWLYGTEPTE SEQ ID NO: 239 618-B11,SETRPTQADYLHCSGPPTFECFWYGTEPTE SEQ ID NO: 240 617-F01,SETRPTGDGNWHCNGPPTFECWRFGTEPTE SEQ ID NO: 241 617-B01,SETRPTEASNYHCIGPPTFECFWYGTEPTE SEQ ID NO: 242 616-G12,SETRPTEAGDWLCKGPPTFECWWQVTDPTE SEQ ID NO: 243 620-G01,SETRPTEAGSWHCNGPPTFECWWYSSDPTE SEQ ID NO: 244 620-C10,SETRPTEDGGWRCSGPPTFECWWYGTEPTE SEQ ID NO: 245 619-G09,SETRPTEAGRIECKGPPWFSCVIYGTEPTE SEQ ID NO: 246 619-F06,SETRPTGGGSWNCSGPPTFECWWYGTEPTE SEQ ID NO: 247 618-C03,SETRPTEAGSLYCSGPPTFECWWYITHPTE SEQ ID NO: 248 617-F02,SETRPTEAGRWHCSGPPRFECWWYDTEPTE SEQ ID NO: 249 616-H01,SETRPTEYGSWHCSGPPTFECWYHGTEPTE SEQ ID NO: 250 618-D01,SETRPTEAGNWHCSGPPSFECWWYATEPTE SEQ ID NO: 251 617-F03,SETRPTEQGSWHCKGPPTFECWSYGTEPTE SEQ ID NO: 252 616-H03,SETRPTDAANYHCSGPPTFECWWYGTEPTE SEQ ID NO: 253 616-G02,SETRPTEAGSWYCSGPPMFECWWLAEEPTE SEQ ID NO: 254 620-G09,SETRPTEAGGWYCSGPPAFECWWYATEPTE SEQ ID NO: 255 620-D12,SETRPTEAGIWSCSGPPTFECWWYESSPTE SEQ ID NO: 256 619-A09,SETRPTEEGLRVCSGPPTFECWWYGTEPTE SEQ ID NO: 257 618-D06,SETRPTEAGSWLCFGPPTFECWSFGTEPTE SEQ ID NO: 258 617-H12,SETRPTVAGSWDCSGPPTFECWWYGTEPTE SEQ ID NO: 259 616-H05,SETRPTKADNWHCSGPPTFECWWYGTEPTE SEQ ID NO: 260 619-H10,SETRPTEAGIVYCSGPPTFECWWFGTEPTE SEQ ID NO: 261 619-D03,SETRPTEAGYWHCLGPPTFECWWYVKEPTE SEQ ID NO: 262 618-D12,SETRPTEPGLLHCSGPPTFECWWYGTEPTE SEQ ID NO: 263 620-E04,SETRPTEASSWYCSGPPSFECWWYGTEPTE SEQ ID NO: 264 620-A05,SETRPTEAGSWHCLGPPTFECWWYVKEPTE SEQ ID NO: 265 619-D04,SETRPTEAGIILCKGPPWFSCDIYDTGPTE SEQ ID NO: 266 618-A11,SETRPTAAGNWHCSGPPTFECWAYGTEPTE SEQ ID NO: 267 617-D07,SETRPTVGGSWYCSGPPTFECWSYGTEPTE SEQ ID NO: 268 627-A10,SETRPTEDGWLDCKGPPTFECWWYGTEPTE SEQ ID NO: 269 626-H02,SETRPTEDGNWHCSGPPTFECWSYGTEPTE SEQ ID NO: 270 626-F06,SETRPTEAGSWHCSGPPTFECWYYWPEPTE SEQ ID NO: 271 624-D02,SETRPTEAGSLYCSGPPMFECWWYDWYPTE SEQ ID NO: 272 622-D09,SETRPTEAGGWYCMGPPAFECWWYASEPTE SEQ ID NO: 273 621-F11,SETRPTNAGSWYCSGPPTFECWWYGTEPTE SEQ ID NO: 274 621-B11,SETRPTEASRWHCNGPPTFECWWYGTEPTE SEQ ID NO: 275 627-B03,SETRPTEAGSFVCSGPPTFECWWYNTGPTE SEQ ID NO: 276 626-H03,SETRPTEAGSWHCSGPPTFECWSYGTEPTE SEQ ID NO: 277 626-F07,SETRPTESDIWLCSGPPTFECWWYGTEPTE SEQ ID NO: 278 626-D02,SETRPTDADPWHCSGPPTFECWWFGTEPTE SEQ ID NO: 279 625-B03,SETRPTEAGVVLCSGPPTFECWWYDTEPTE SEQ ID NO: 280 622-D10,SETRPTEVGSVHCSGPPTFECWWFGTEPTE SEQ ID NO: 281 621-G02,SETRPTEAGRWLCSGPPTFECWEYDTEPTE SEQ ID NO: 282 621-E04,SETRPTDAGWLQCSGPPTFECWWYGTEPTE SEQ ID NO: 283 621-B12,SETRPTEASRRHCNGPPTFECWRYGTEPTE SEQ ID NO: 284 626-H04,SETRPTEAGRWYCSGPPTFECWLFVEEPTE SEQ ID NO: 285 626-F11,SETRPTAADSWQCSGPPTFECWSFGTEPTE SEQ ID NO: 286 626-D03,SETRPTEAGSWHCGGPPTFECWMYVTEPTE SEQ ID NO: 287 626-A02,SETRPTDDGSWYCSGPPTFECWWYGTEPTE SEQ ID NO: 288 623-E07,SETRPTEAGYWHCLGPPTFECWWYDMEPTE SEQ ID NO: 289 622-G09,SETRPTEAGILRCSGPPTFECWYYETEPTE SEQ ID NO: 290 622-E05,SETRPTEDVSVHCAGPPTFECWLYGTEPTE SEQ ID NO: 291 622-B12,SETRPTEEGVFQCVGPPTFECWWYGTEPTE SEQ ID NO: 292 621-G07,SETRPTEDGGFFCSGPPTFECWWYGTEPTE SEQ ID NO: 293 621-E07,SETRPTEPGSWHCSGPPTFECWWYGTEPTE SEQ ID NO: 294 621-C01,SETRPTEAGSWHCSGPPTFECWWYDRAPTE SEQ ID NO: 295 626-A05,SETRPTEAGTWYCSGPPTFECWYYATEPTE SEQ ID NO: 296 623-G02,SETRPTEAGSLYCSGPPAFECYWYGTVPTE SEQ ID NO: 297 622-H11,SETRPTDPGVLHCSGPPTFECWWFGTEPTE SEQ ID NO: 298 622-C04,SETRPTEAGTWYCLGPPTFECWSFWQDPTE SEQ ID NO: 299 621-G11,SETRPTEAGRWGCSGPPTFECWWYVAEPTE SEQ ID NO: 300 621-C07,SETRPTEAGIWHCAGPPTFICWLYETEPTE SEQ ID NO: 301 627-C03,SETRPTEAGSWHCSGPPSFECWQYSTEPTE SEQ ID NO: 302 626-D12,SETRPTEAGSWQCSGPPTFECWVYETEPTE SEQ ID NO: 303 626-A06,SETRPTEAGSWYCSGPPTFECWWYDVGPTE SEQ ID NO: 304 623-H02,SETRPTDEVSWECRGPPTFECWWYGTEPTE SEQ ID NO: 305 623-B05,SETRPTEGGSWVCSGPPTFECWWYGTEPTE SEQ ID NO: 306 622-E10,SETRPTEYGSWYCSGPPTFECWWLGTEPTE SEQ ID NO: 307 622-C06,SETRPTEAGVWLCSGPPTFECWWYDTDPTE SEQ ID NO: 308 621-H03,SETRPTMAGSYYCSGPPTFECWVYGTEPTE SEQ ID NO: 309 621-E11,SETRPTEAGYVQCYGPPSFVCHPMVPDPTE SEQ ID NO: 310 621-C08,SETRPTEDGFVLCKGPPWFSCEMYGTEPTE SEQ ID NO: 311 627-C04,SETRPTEAGGWNCSGPPTFECWWYVTEPTE SEQ ID NO: 312 626-A07,SETRPTEDGSWECFGPPTFECWSYGTEPTE SEQ ID NO: 313 623-H08,SETRPTDAVSYVCKGPPTFECWWYGTEPTE SEQ ID NO: 314 622-F05,SETRPTEARSWHCSGPPTFECWWYGTEPTE SEQ ID NO: 315 627-A04,SETRPTASVSWHCSGPPTFECWSYGTEPTE SEQ ID NO: 316 626-G05,SETRPTEAGSWYCSGPPTFECWYYDMDPTE SEQ ID NO: 317 623-H11,SETRPTEAGSWLCSGPPTFECWWFGTEPTE SEQ ID NO: 318 622-F11,SETRPTGDGSWYCSGPPTFECWWLGTEPTE SEQ ID NO: 319 621-F03,SETRPTEAGSWYCSGPPTFECWWYFLDPTE SEQ ID NO: 320 626-F01,SETRPTEAGGWYCSGPPTFECWWFATEPTE SEQ ID NO: 321 621-F04,SETRPTEAGDLDCLGPPTFICRIYGTEPTE SEQ ID NO: 322 630-F06,SETRPTEAGSWQCVGPPTFECWSFGTEPTE SEQ ID NO: 323 630-A03,SETRPTEADSWYCSGPPTFECWLFGTEPTE SEQ ID NO: 324 629-F10,SETRPTQADSWYCSGPPTFECWWWGTEPTE SEQ ID NO: 325 629-D11,SETRPTEAFSWDCSGPPTFECWWFGTEPTE SEQ ID NO: 326 629-B06,SETRPTEAGSWQCSGPPVFECWWYDTEPTE SEQ ID NO: 327 628-H01,SETRPTEAGNVQCSGPPTFECWWFDTEPTE SEQ ID NO: 328 628-F03,SETRPTEAGSVVCSGPPRFECWAFVTEPTE SEQ ID NO: 329 627-G02,SETRPTEDGTLHCSGPPTFACWWYGTEPTE SEQ ID NO: 330 629-E01,SETRPTDAEVWVCNGPPTFECWWYGTEPTE SEQ ID NO: 331 628-H09,SETRPTEDVTFHCSGPPTFECWLYGTEPTE SEQ ID NO: 332 628-A05,SETRPTSDFDWHCKGPPTFECWSYGTEPTE SEQ ID NO: 333 627-G04,SETRPTEADSWYCSGPPTFECWWYVPEPTE SEQ ID NO: 334 630-A05,SETRPTDDGNWYCSGPPTFECWWYGTEPTE SEQ ID NO: 335 629-E03,SETRPTEAGSWYCSGPPTFECWRYDTDPTE SEQ ID NO: 336 629-C02,SETRPTEAGPWSCSGPPTFECWWFDTEPTE SEQ ID NO: 337 628-H10,SETRPTEAGMFLCSGPPAFECWWYDTEPTE SEQ ID NO: 338 628-F12,SETRPTEAGSLYCSGPPTFECWLYDVEPTE SEQ ID NO: 339 627-D12,SETRPTEAGQWNCSGPPTFECWWYDIEPTE SEQ ID NO: 340 630-G02,SETRPTEAGSWYCSGPPTFECWWFETEPTE SEQ ID NO: 341 629-E06,SETRPTEAGSFVCSGPPTFECWGYVTEPTE SEQ ID NO: 342 628-D07,SETRPTQDGTWFCSGPPTFECWWYGTEPTE SEQ ID NO: 343 627-E06,SETRPTEGDSWHCAGPPTFECWWYGTEPTE SEQ ID NO: 344 629-E07,SETRPTEAGSWSCSGPPTFECWSYGTEPTE SEQ ID NO: 345 629-C11,SETRPTEAGRIQCSGPPTFECWWYDEEPTE SEQ ID NO: 346 629-A03,SETRPTEAGTIVCKGPPWFSCEIYETEPTE SEQ ID NO: 347 628-A12,SETRPTEAGDWYCSGPPAFECWEYLGEPTE SEQ ID NO: 348 627-E08,SETRPTEAGSWFCSGPPSFECWSYVTEPTE SEQ ID NO: 349 629-E08,SETRPTEAGSWHCSGPPAFECWWYDNEPTE SEQ ID NO: 350 629-B02,SETRPTEAGRWTCSGPPTFECWWYVSDPTE SEQ ID NO: 351 628-E06,SETRPTEAGEWYCGGPPTFECWWFDTAPTE SEQ ID NO: 352 627-G09,SETRPTEAGSWHCSGPPSFECWWFDTGPTE SEQ ID NO: 353 631-A11,SETRPTEAGSFICSGPPTFECWWYGTEPTE SEQ ID NO: 354 630-C10,SETRPTEDVRWYCSGPPTFECWWFGTEPTE SEQ ID NO: 355 628-B08,SETRPTEAGSWYCSGPPTFECWWYVPEPTE SEQ ID NO: 356 629-F03,SETRPTEAGNWLCSGPPAFECWWFVAEPTE SEQ ID NO: 357 632-A09,SETRPTEAGSWYCSGPPTFECWWYGTEPTE SEQ ID NO: 358 632-G07,SETRPTEAGDWLCAGPPTFECWWWGTDPTE SEQ ID NO: 359 631-F12,SETRPTEAGSWHCVGPPTFECWWFDTEPTE SEQ ID NO: 360 633-A02,SETRPTEAGEWSCSGPPTFECWWWDMEPTE SEQ ID NO: 361 633-B06,SETRPTYYVSWYCSGPPTFECWSYGTEPTE SEQ ID NO: 362 632-D11,SETRPTEDGSWYCSGPPTFECWWYGTEPTE SEQ ID NO: 363 631-D10,SETRPTEDGTWYCSGPPTFECWWYGTEPTE SEQ ID NO: 364 633-F09,SETRPTETDSWVCSGPPTFECWWYGTEPTE Consensus Motif#1:G-X1-X2-X3-C-X4-G-P-P-X5-F-X6-C-X7-X8- X9-X10-X11-X12-P-T-E, where: X1is any amino acid other than C, preferably S, R, I, D, or N; X2 is anyamino acid other than C, preferably W, L, F, V, or I; X3 is any aminoacid other than C, preferably H, Y, L, Q, N, or V; X4 is any amino acidother than C, preferably S, K, or L; X5 is any amino acid other than C,preferably T, S, A, or W; X6 is any amino acid other than C, preferablyE or S; X7 is any amino acid other than C, preferably W; X8 is any aminoacid other than C, preferably W, S, or L; X9 is any amino acid otherthan C, preferably Y or F; X10 is any amino acid other than C,preferably D, G, V, or E; X11 is any amino acid other than C, preferablyT, P, M, or S; and X12 is any amino acid other than C, preferably E orG. Motif#2: T-X1-X2-X3-X4-X5-X6-C-X7-G-P-P-X8-F-X9-C-X10-X11- X12-G,where: X1 is any amino acid other than C, preferably E, D, or V; X2 isany amino acid other than C, preferably A, D, G, S, or V; X3 is anyamino acid other than C, preferably G, V, D, or S; X4 is any amino acidother than C, preferably S, N, R, T, or G; X5 is any amino acid otherthan C, preferably W; X6 is any amino acid other than C, preferably H orQ; X7 is any amino acid other than C, preferably S, N, or K; X8 is anyamino acid other than C, preferably T; X9 is any amino acid other thanC, preferably E; X10 is any amino acid other than C, preferably W; X11is any amino acid other than C, preferably W or S; and X12 is any aminoacid other than C, preferably Y or F. CLASS V TN10: SEQ ID NO: 365545-C02, GSWRFCGGEYSFQVCQDVAP SEQ ID NO: 366 546-E02,GSHHTCLDGFAGWRCTEVAP SEQ ID NO: 367 545-C11, GSFAPCGWPSFAIDCIAEAP SEQ IDNO: 368 549-G01, GSTKVCHEKWNQLFCHNQAP SEQ ID NO: 369 548-F07,GSPEMCMMFPFLYPCNHHAP SEQ ID NO: 370 551-H10, GSFFPCWRIDRFGYCHANAPConsensus Motif: S-X1-X2-X3-C-X4-X5-X6-X7-X8-X9-X10-X11-C-X12-X13-X14-A-P, where X1 is one of W, H, F, T, or P; X2 is one of R,H, A, K, E, or F; X3 is one of F, T, P, V, or M; X4 is one of F, L, H,M, or W; X5 is one of G, D, W, E, M, or R; X6 is one of E, G, P, K, F,or I; X7 is one of Y, F, S, W, P, or D; X8 is one of S, A, F, N, or R;X9 is one of F, G, A, Q, or L; X10 is one of Q, W, I, L, Y, or G; X11 isone of V, R, D, F, P, or Y; X12 is one of Q, T, I, H, or N; X13 is oneof D, E, A, N, or H; and X14 is one of V, E, Q, H, or N CLASS VI TN11#1: SEQ ID NO: 371 443-H10, GSQQICDRKEYRFQACLSDAP SEQ ID NO: 372557-A12, GSTMSCWRWGRDAYSCNQMAP SEQ ID NO: 373 465-A03,GSSQICAVYLDDTHNCERHAP SEQ ID NO: 374 446-E12, GSSHCNQMITPWQNCGMRAP SEQID NO: 375 445-E06, GSSARCDELINDFHSCLVMAP SEQ ID NO: 376 452-A03,GSRFHCWQGDLMQTYCMPMAP SEQ ID NO: 377 465-C06, GSQNNCEYGSRGSSFCLAMAP SEQID NO: 378 441-H01, GSMNMCDTTDEISPTCHPSAP SEQ ID NO: 379 443-D04,GSMLGCLFEHQNKYDCYVLAP SEQ ID NO: 380 445-G12, GSLYRCLGEASPTPPCAYEAP SEQID NO: 381 442-E03, GSGMGCHQVNISTGDCAEDAP SEQ ID NO: 382 453-A05,GSGDPCSPGPSINGHCSVMAP SEQ ID NO: 383 445-E07, GSFWNCTTDLGAMSDCGFFAP SEQID NO: 384 451-B12, GSFTACNKTSTTRQPCNPYAP SEQ ID NO: 385 465-B07,GSELFCFYHHQGYEGCDVLAP SEQ ID NO: 386 451-C06, GSDMNCTVLAQDQIFCFREAP SEQID NO: 387 445-E11, GSAGWCYTMNYVDQLCTYMAP Consensus Motif:S-X1-X2-X3-C-X4-X5-X6-X7-X8-X9-X10-X11- X12-C-X13-X14-X15-A-P, where X1is any amino acid other than C, preferably S, F, G, M, or Q; X2 is anyamino acid other than C, preferably M, L, N, or Q; X3 is any amino acidother than C, preferably N, G, H, I, or R; X4 is any amino acid otherthan C, preferably D, L, N, T, or W; X5 is any amino acid other than C,preferably Q, T, R, V, or Y; X6 is any amino acid other than C,preferably G, E, L, M, or T; X7 is any amino acid other than C,preferably A, D, H, I, L, N, or S; X8 is any amino acid other than C,preferably Q, R, S, T, or Y; X9 is any amino acid other than C,preferably D, G, I, or P; X10 is any amino acid other than C, preferablyT, F, or Q; X11 is any amino acid other than C, preferably Q, F, H, P,S, or Y; X12 is any amino acid other than C, preferably D, F, N, P, orS; X13 is any amino acid other than C, preferably L, A, G, N, or S; X14is any amino acid other than C, preferably V, P, R, or Y; and X15 is anyamino acid other than C, preferably M, D, E, or L. CLASS VII TN11 #2 SEQID NO: 388 593-G11, SETRPTEAGMCACRGPPAFVCQWYGSEPTE SEQ ID NO: 389631-E12, SETRPTEAGSCHCSGPPTFECWSYVTEPTE CLASS VIII TN12: SEQ ID NO: 390546-G02, GDYDYCDFDLETYIPECHSYDP SEQ ID NO: 391 333-C03,GDDFHCEFIDDYQSEICYFNDP SEQ ID NO: 392 549-G05, GDLLVCKFDDKFWTETCEWADPSEQ ID NO: 393 546-B01, GDSYNCSWDSKTFEVTCLYADP SEQ ID NO: 394 551-D02,GDASWCDENSPAAWFYCELWDP SEQ ID NO: 395 334-F05, GDLLGCGYQEKGGEYKCRFNDPSEQ ID NO: 396 330-G02, GDPWWCFEKDSFIPFACWHHDP SEQ ID NO: 397 316-F08,GDYYQCQFSKDMYSERCWPYDP SEQ ID NO: 398 332-H09, GDNRFCSWVYNVDDWWCVDNDPSEQ ID NO: 399 545-H12, GDYSECFFEPDSFEVKCYDRDP SEQ ID NO: 400 548-G05,GDYRMCQISDMWGNYECSSDDP SEQ ID NO: 401 547-C09, GDPDECQLNRETFEVWCPWHDPSEQ ID NO: 402 545-F04, GDHRKCEISAKTHEVTCYDNDP SEQ ID NO: 403 552-F06,GDHLTCEFRDDGWKEHCWWSDP SEQ ID NO: 404 531-E11, GDASMCYDGLALRWDQCWPHDPConsensus Motif: D-X1-X2-X3-C-X4-X5-X-6-X7-X8-X9-X10-X11-X12-X13-C-X14-X15-X16-D-P, where X1 is any amino acid other than C,preferably Y, A, H, L, or P; X2 is any amino acid other than C,preferably L, R, S, D, or Y; X3 is any amino acid other than C,preferably E, M, or W; X4 is any amino acid other than C, preferably E,Q, D, F, or S; X5 is any amino acid other than C, preferably F, I, W, orE; X6 is any amino acid other than C, preferably D, S, or N; X7 is anyamino acid other than C, preferably D, S, or L; X8 is any amino acidother than C, preferably D, K, or E; X9 is any amino acid other than C,preferably T, F, or G; X10 is any amino acid other than C, preferably F,W, Y, or G; X11 is any amino acid other than C, preferably E, S, or W;X12 is any amino acid other than C, preferably E, V, F, or Y; X13 is anyamino acid other than C, preferably T, E, K, or V; X14 is any amino acidother than C, preferably W or E; X15 is any amino acid other than C,preferably D, W, F, P, or S; and X16 is any amino acid other than C,preferably N, I, or A. CLASS IX TN9 #3: SEQ ID NO: 405 606-B08,SETRPTEAGSCHCSGPPTFQCWCYEVEPTE SEQ ID NO: 406 602-G12,SETRPTEAGSCHCSGPPTFECWCYGTEPTE SEQ ID NO: 407 603-E09,SETRPTGESDCHCSGPPTFECYCYGTEPTE SEQ ID NO: 408 606-C12,SETRPTESGNCYCSGPPWFECWCYGTEPTE SEQ ID NO: 409 603-H03,SETRPTEAGACRCSGPPTFECYCYDMAPTE SEQ ID NO: 410 604-G01,SETRPTEAGSCYCSGPPRFECWCYETEPTE SEQ ID NO: 411 602-G04,SETRPTEAGSCHCSGPPSFECWCFGTEPTE SEQ ID NO: 412 611-G11,SETRPTVSVSCSCGGPPTFECWCFGTEPTE SEQ ID NO: 413 611-F02,SETRPTEAGSCHCNGPPTFECFCFGTEPTE SEQ ID NO: 414 610-G02,SETRPTEAGSCYCGGPPSFECWCYGTEPTE SEQ ID NO: 415 614-E08,SETRPTEAGSCHCSGPPTFECWCYGSNPTE SEQ ID NO: 416 615-A01,SETRPTEAGSCHCSGPPAFECWCYRAEPTE SEQ ID NO: 417 617-H02,SETRPTEAGSCDCSGPPTFECWCFGTEPTE SEQ ID NO: 418 616-F12,SETRPTEAGKCHCGGPPSFECWCYATEPTE SEQ ID NO: 419 620-G06,SETRPTEAGKCHCSGPPTFECTCYHTDPTE SEQ ID NO: 420 627-B04,SETRPTEAGFCQCSGPPAFECWCYDTEPTE SEQ ID NO: 421 627-B06,SETRPTEAVSCECKGPPTFECWCFGTEPTE SEQ ID NO: 422 626-H05,SETRPTEAGDCHCSGPPTFECWCYGTEPTE SEQ ID NO: 423 626-D11,SETRPTEAGACDCIGPPTFECWCYDTYPTE SEQ ID NO: 424 626-E05,SETRPTEAGNCLCSGPPTFECACYHSEPTE SEQ ID NO: 425 621-D01,SETRPTEAGSCHCSGPPTFQCWCYSTEPTE SEQ ID NO: 426 622-A10,SETRPTEAGICHCSGPPTFECWCYATEPTE SEQ ID NO: 427 630-D09,SETRPTEEGSCHCSGPPTFECWCFGTEPTE SEQ ID NO: 428 628-D01,SETRPTEAGICNCSGPPTFECWCYSMGPTE SEQ ID NO: 429 628-F11,SETRPTQGGNCHCSGPPTFECWCYGTEPTE SEQ ID NO: 430 628-D04,SETRPTEAGSCNCSGPPTFECYCYTLDPTE SEQ ID NO: 431 630-G01,SETRPTDNGSCQCSGPPTFECWCFGTEPTE SEQ ID NO: 432 627-G06,SETRPTESGSCHCSGPPTFECWCYGTEPTE SEQ ID NO: 433 630-G05,SETRPTEAGSCNCSGPPSFECWCYVTEPTE SEQ ID NO: 434 630-C03,SETRPTEGGSCYCGGPPTFECWCYGTEPTE SEQ ID NO: 435 627-G07,SETRPTEAGRCHCSGPPTFECWCYVQEPTE SEQ ID NO: 436 630-H10,SETRPTESGSCLCSGPPQFECWCYGTEPTE SEQ ID NO: 437 628-B01,SETRPTETDSCHCIGPPTFECWCYGTEPTE SEQ ID NO: 438 630-F01,SETRPTEAGFCRCSGPPTFECWCYDTEPTE SEQ ID NO: 439 629-D01,SETRPTEHGSCNCYGPPTFECWCYGTEPTE SEQ ID NO: 440 633-G02,SETRPTALGGCLCSGPPTFECWCYGTEPTE SEQ ID NO: 441 631-F07,SETRPTEGGSCECSGPPTFECWCYGTEPTE SEQ ID NO: 442 633-G08,SETRPTEEGSCHCSGPPAFECWCYGTEPTE SEQ ID NO: 443 632-H07,SETRPTEAGTCYCSGPPTFECWCYGTEPTE SEQ ID NO: 444 631-D03,SETRPTEDGSCHCSGPPRFECWCYGTEPTE SEQ ID NO: 445 633-G12,SETRPTEAGSCHCSGPPTFECWCYSTEPTE SEQ ID NO: 446 633-H03,SETRPTEAGSCYCSGPPTFECWCYAEEPTE SEQ ID NO: 447 632-F05,SETRPTEAGSCHCSGPPTFECWCFEPEPTE Motif13-1G-X1-C-X2-C-X3-G-P-P-X4-F-X5-C-X6-C-X7-X8-X9-X10- P, where X1 is anyamino acid other than C, preferably S; X2 is any amino acid other thanC, preferably H, Y, or N; X3 is any amino acid other than C, preferablyS or G; X4 is any amino acid other than C, preferably T; X5 is any aminoacid other than C, preferably E; X6 is any amino acid other than C,preferably W; X7 is any amino acid other than C, preferably Y; X8 is anyamino acid other than C, preferably G, D, A, E, or S; X9 is any aminoacid other than C, preferably T or S; and X10 is any amino acid otherthan C, preferably E or D. Motif13-2 isT-X1-X2-X3-X4-C-X5-C-X6-G-P-P-X7-F-E-C-X8-C- X9-G where: X1 is any aminoacid other than C, preferably E; X2 is any amino acid other than C,preferably A, S, E, or G; X3 is any amino acid other than C, preferablyG; X4 is any amino acid other than C, preferably S; X5 is any amino acidother than C, preferably H; X6 is any amino acid other than C,preferably S; X7 is any amino acid other than C, preferably T; X8 is anyamino acid other than C, preferably W, Y, or F; and X9 is any amino acidother than C, preferably Y or F. CLASS X SEQ ID NO: 448 606-E11,SEYPTWVSKEFHECAGELVAMQGGSGTE CLASS XI Linear #1: SEQ ID NO: 449 525-A07,AQQASRFTFTDGDSYWWFEDF SEQ ID NO: 450 528-F05, AQIQGIQKTEQGEFYWFNWFPA SEQID NO: 451 524-E09, AQREVEEPYWYLDFLSSWRMHE SEQ ID NO: 452 96-H12,AQRPEAHYKLAMSYPIIPRTKT SEQ ID NO: 453 118-A08, AQRWSSPGMSQSFVLEWKWNDNSEQ ID NO: 454 94-E08, AQYDTWVFQFIHEVPGELVAMQ SEQ ID NO: 455 119-F06,AQMYQTPDGVIGKFVDWMFN SEQ ID NO: 456 95-A11, AQVGSPMLPSWFSFEANWSS SEQ IDNO: 457 94-H04, AQNAVVPPPMLWSIYWDYGREG SEQ ID NO: 458 94-F07,AQPYYELQDADMLLVVALLSTG SEQ ID NO: 459 103-G08, AQVGTAEAIMFSDVEDTGVHKFSEQ ID NO: 460 118-C07, AQFPLEFDVPNFSYHWLVSFNP SEQ ID NO: 461 104-C09,AQDLKPWTAGWEPPWLWTDRGP SEQ ID NO: 462 117-F08, AQHQYGQMMVLHIQYDMGEFIPSEQ ID NO: 463 76-D09, AQSPYIFPIDDSGRQIFVIQWG SEQ ID NO: 464 93-C08,AQVPDWLSAVVIEKLIEYGMMV SEQ ID NO: 465 92-B05, AQFDRYWHFAWMDVSFSSGQSG SEQID NO: 466 116-H02, AQKETWEFFDIVYGSGWKFNSP SEQ ID NO: 467 02-B08,AQHSVQRQMDVWMPVQFMAGFT SEQ ID NO: 468 117-F03, AQEWQTWTWNMIEVISENKTP SEQID NO: 469 127-A07, AQGFELWVDHTRNFFIAISP SEQ ID NO: 470 94-B08,AQAYEWWADESIFNHGYYWGHQ SEQ ID NO: 471 115-G02, AQDPGFSKHSMGHGYPSKMNWGSEQ ID NO: 472 130-E10, AQEWEREYFVDGFWGSWFGIPH SEQ ID NO: 473 136-D01,AQMGHHWDVQWDYKLFHVARGD SEQ ID NO: 474 15-D02, AQELFQILEKQMWSDFMEWATP SEQID NO: 475 79-B02, AQHWDYDSGSDFWFPVFFLEHH SEQ ID NO: 476 94-A06,AQHGYLSPLKQYQMSHVEFWTY SEQ ID NO: 477 94-G02, AQFSGLVMYGRTHEVQWTFGSM SEQID NO: 478 75-B12, AQAEWVITSEEFYWKMADFGPP SEQ ID NO: 479 117-F04,AQWPHDGLVHWGEVIMLRF SEQ ID NO: 480 151-B08, AQWNQWDEFMWFLNPPPIGLMW SEQID NO: 481 117-E09, AQDNTADQMFNGFHVLAMYMV SEQ ID NO: 482 93-B10,AQSDHDHAHWGVKHWPFRRYQ SEQ ID NO: 483 98-F05, AQLFQYLWHDDPQGAFFQLSMW SEQID NO: 484 118-B12, AQHVVTLTLIQMPFAFNFEPRM SEQ ID NO: 485 27-D10,AQVGESLDDGWTFFSDKWFDFF SEQ ID NO: 486 122-D07, AQFMYEKEHYVMSISLPGLWFYSEQ ID NO: 487 149-E06, AQHMDPAEWDWFIRIYSPVVNP SEQ ID NO: 488 166-H04,AQMWHRVHDPGYTFEVTWLWDN SEQ ID NO: 489 96-D06, AQWNWDMGFMWTTDSAQVQPSM SEQID NO: 490 103-C04, AQKTWFLEADLFQMFQEVTWQF SEQ ID NO: 491 527-E08,AQWGAVDNDWYDWEMEQIWMFE SEQ ID NO: 492 524-H02, AQVEDMATVHFKFNPATHEVIWSEQ ID NO: 493 523-A04, AQRDYLFYWNDGSYQPWQVFVG SEQ ID NO: 494 524-D07,AQQWMFQIHQSMAWPYEWIDSY SEQ ID NO: 495 522-H03, AQGIAWQLEWSYMPQSPPSFDRSEQ ID NO: 496 527-A10, AQGGRYPFYDTDWFKWEMYVL CLASS XII Linear #2 SEQ IDNO: 497 594-F01, SEEDTWLFWQIIEVPVGQVLMQGGSGTE SEQ ID NO: 498 592-E11,SEYDTLLFQRTGEVVGKLGSMQGGSGTE SEQ ID NO: 499 591-G09,SEYDTWVFQFMLEVPGSWMARLGGSGTE SEQ ID NO: 500 601-G11,SEYDTWIFQFYREVPGVPGAMQGGSGTE SEQ ID NO: 501 592-G01,SEVDTGVQLLTHEGPGELVAMQGGSGTE SEQ ID NO: 502 591-H01,SESDTWVFQLIHEVPASVVAMQGGSGTE SEQ ID NO: 503 592-G05,SEYDTWVFQFRHGVKAQLVAMRGGSGTE SEQ ID NO: 504 606-D12,SEYDSRVFQYAPEVAGQVEAMQGGSGTE SEQ ID NO: 505 592-B01,SEDESRVVQFQHEVSGELVAMQGGSGTE SEQ ID NO: 506 591-A06,SEQDTFVFMYNGEVSGDMVAMQGGSGTE SEQ ID NO: 507 588-H01,SEYDTWVFQFRRQVPGVLETMLGGSGTE SEQ ID NO: 508 589-A01,SEQETLVFAVIDGDPGELVAMQGGSGTE SEQ ID NO: 509 619-F10,SEYDTWVFQFIHVARGEMEGTLGGSGTE SEQ ID NO: 510 592-B01,SEDESRVVQFQHEVSGELVAMQGGSGTE SEQ ID NO: 511 591-A06,SEQDTFVFMYNGEVSGDMVAMQGGSGTE

TABLE 7 Protein WC HGF 100 HGF 500 SEQ ID NO: Isolate ELISA ELISA ng/mLng/mL CLASS I SEQ ID 571-C05 4.9 1.30 102%  74% NO: 001 SEQ ID 465-A064.4 1.33 56% 32% NO: 002 SEQ ID 465-D09 3.2 1.30 90% 70% NO: 003 SEQ ID569-H10 3.4 1.27 98% 83% NO: 004 SEQ ID 470-E11 3.5 1.33 55% 127%  NO:005 SEQ ID 452-F01 3.2 1.33 117%  110%  NO: 006 SEQ ID 569-C03 3.4 1.3095% 89% NO: 007 SEQ ID 574-H03 3.2 1.27 88% 18% NO: 008 SEQ ID 567-C083.8 1.27 85% 94% NO: 009 SEQ ID 561-C08 3.0 1.37 92% 96% NO: 010 CLASSII SEQ ID 573-F04 5.6 1.30 76% 71% NO: 011 SEQ ID 570-E07 4.5 1.27 81%71% NO: 012 SEQ ID 456-E04 3.9 1.40 82% 81% NO: 013 SEQ ID 434-E12 4.81.33 117%  41% NO: 014 SEQ ID 489-A04 4.3 1.33 30% 13% NO: 015 SEQ ID484-D08 4.1 1.33 105%  90% NO: 016 SEQ ID 482-D02 3.9 1.37 66% 44% NO:017 SEQ ID 437-A09 3.9 1.13 89% 78% NO: 018 SEQ ID 352-E04 3.9 1.37 88%74% NO: 019 SEQ ID 376-E05 3.7 1.37 122%  121%  NO: 020 SEQ ID 482-A123.5 1.37 98% 79% NO: 021 SEQ ID 423-C11 3.4 1.40 132%  75% NO: 022 SEQID 499-C09 3.2 1.33 91% 70% NO: 023 SEQ ID 457-A09 14.5 1.30 27% 67% NO:024 SEQ ID 573-E07 3.2 1.37 77% 82% NO: 025 SEQ ID 465-F08 3.8 1.30 68%116%  NO: 026 SEQ ID 465-E09 3.6 1.30 60% 77% NO: 027 SEQ ID 444-B08 3.61.43 111%  93% NO: 028 SEQ ID 465-E11 4.3 1.23 33% 124%  NO: 029 SEQ ID465-D12 3.2 1.27 34%  0% NO: 030 SEQ ID 470-A02 3.2 1.30 78% 62% NO: 031SEQ ID 465-C01 3.2 1.27 267%  23% NO: 032 SEQ ID 448-H02 3.8 1.43 113% 92% NO: 033 SEQ ID 465-D01 3.3 1.30 235%  134%  NO: 034 SEQ ID 571-C113.5 1.23 107%  72% NO: 035 SEQ ID 465-B11 3.6 1.27 97% 89% NO: 036 SEQID 442-E08 4.1 1.43 81% 75% NO: 037 SEQ ID 465-C11 3.1 1.30 41%  4% NO:038 SEQ ID 465-F10 3.7 1.33 61% 42% NO: 039 SEQ ID 471-A11 3.0 1.37 85%80% NO: 040 SEQ ID 465-C07 3.1 1.27 102%  138%  NO: 041 SEQ ID 465-D043.1 1.23 77% 31% NO: 042 SEQ ID 445-E04 4.2 1.37 127%  102%  NO: 043 SEQID 465-B06 4.1 1.23 89% 57% NO: 044 SEQ ID 470-C02 3.9 1.33 340%  227% NO: 045 SEQ ID 458-B05 4.5 1.33 201%  247%  NO: 046 SEQ ID 545-E08 4.71.30 81% 57% NO: 047 CLASS III SEQ ID 325-H05 15.9 1.47 41% 32% NO: 048SEQ ID 330-F05 13.8 1.33 51% 27% NO: 049 SEQ ID 333-F09 14.8 1.43 52%32% NO: 050 SEQ ID 336-G04 5.4 1.33 46% 23% NO: 051 SEQ ID 334-G06 8.01.30 56% 43% NO: 052 SEQ ID 330-B07 18.1 1.27 58% 40% NO: 053 SEQ ID330-C10 13.4 1.33 48% 25% NO: 054 SEQ ID 331-G04 18.3 1.47 56% 36% NO:055 SEQ ID 548-F06 14.3 1.23 76% 18% NO: 056 SEQ ID 538-F08 12.3 1.2355% 43% NO: 057 SEQ ID 547-H07 15.9 1.17 60% 45% NO: 058 SEQ ID 323-A1121.2 1.43 41% 18% NO: 059 SEQ ID 333-H03 8.1 1.43 55% 37% NO: 060 SEQ ID329-D02 3.2 1.27 53% 31% NO: 061 SEQ ID 550-C09 10.2 1.40 25% 25% NO:062 SEQ ID 548-E08 5.3 1.27 102%  50% NO: 063 SEQ ID 332-A05 6.0 1.4040% 21% NO: 064 SEQ ID 330-C01 4.7 1.30 58% 43% NO: 065 SEQ ID 545-A0913.5 1.30 44% 22% NO: 066 SEQ ID 334-C08 8.0 1.47 70% 57% NO: 067 SEQ ID333-C05 6.3 1.33 83% 66% NO: 068 SEQ ID 551-B02 9.0 1.30 69% 43% NO: 069SEQ ID 551-G12 3.9 1.37 88% 46% NO: 070 SEQ ID 330-G09 13.5 1.40 42% 26%NO: 071 SEQ ID 331-F01 12.6 1.47 77% 73% NO: 072 SEQ ID 274-B07 7.8 1.10342%  296%  NO: 073 SEQ ID 335-D11 6.7 1.37 56% 37% NO: 074 SEQ ID336-D07 5.8 1.33 44% 37% NO: 075 SEQ ID 332-C03 5.7 1.20 37% 95% NO: 076SEQ ID 331-D03 5.5 1.40 64% 55% NO: 077 SEQ ID 331-G06 4.7 1.40 59% 51%NO: 078 SEQ ID 552-G03 10.7 1.27 101%  83% NO: 079 SEQ ID 552-G11 7.41.23 55% 41% NO: 080 SEQ ID 550-G08 9.1 1.40 79% 58% NO: 081 SEQ ID550-G12 14.3 1.43 61% 79% NO: 082 SEQ ID 552-A01 3.9 1.33 76% 81% NO:083 SEQ ID 548-C06 13.0 1.23 94% 77% NO: 084 SEQ ID 545-B12 17.1 1.2751% 42% NO: 085 SEQ ID 549-F06 5.2 1.30 96% 40% NO: 086 SEQ ID 552-F014.8 1.30 56% 37% NO: 087 SEQ ID 547-H12 5.6 1.10 92% 81% NO: 088 SEQ ID550-F11 12.4 1.23 58% 23% NO: 089 SEQ ID 548-D08 19.5 1.23 97% 62% NO:090 SEQ ID 549-D02 8.9 1.27 47% 36% NO: 091 SEQ ID 552-F02 12.3 1.23 60%40% NO: 092 SEQ ID 545-E04 16.3 1.23 48% 17% NO: 093 SEQ ID 545-E05 10.31.27 70% 32% NO: 094 SEQ ID 547-H03 16.2 1.23 109%  53% NO: 095 SEQ ID552-G09 9.7 1.27 98% 68% NO: 096 SEQ ID 550-A08 8.4 1.27 52% 51% NO: 097SEQ ID 550-G07 6.2 1.27 63% 36% NO: 098 SEQ ID 551-A05 4.0 1.30 68% 42%NO: 099 SEQ ID 548-C10 8.4 1.20 69% 57% NO: 100 SEQ ID 465-C10 3.0 1.2795% 71% NO: 101 CLASS V SEQ ID 545-C02 26.3 1.33 54% 31% NO: 365 SEQ ID546-E02 10.4 1.33 74% 54% NO: 366 SEQ ID 545-C11 7.7 1.30 77% 50% NO:367 SEQ ID 549-G01 7.0 1.27 62% 18% NO: 368 SEQ ID 548-F07 27.5 2.43 54%37% NO: 369 SEQ ID 551-H10 13.3 1.87 88% 49% NO: 370 CLASS VI SEQ ID443-H10 3.4 1.40 124%  143%  NO: 371 SEQ ID 557-A12 4.6 1.37 87% 62% NO:372 SEQ ID 465-A03 4.0 1.30 33% 17% NO: 373 SEQ ID 446-E12 3.3 1.37 73%83% NO: 374 SEQ ID 445-E06 4.3 1.33 83% 73% NO: 375 SEQ ID 452-A03 3.01.30 140%  112%  NO: 376 SEQ ID 465-C06 6.4 1.23 184%  104%  NO: 377 SEQID 441-H01 3.6 1.40 91% 69% NO: 378 SEQ ID 443-D04 3.2 1.43 69% 73% NO:379 SEQ ID 445-G12 4.0 1.37 85% 52% NO: 380 SEQ ID 442-E03 3.9 1.43130%  81% NO: 381 SEQ ID 453-A05 4.5 1.33 51% 28% NO: 382 SEQ ID 445-E073.1 1.37 82% 64% NO: 383 SEQ ID 451-B12 3.1 1.37 61% 27% NO: 384 SEQ ID465-B07 4.8 1.27 111%  79% NO: 385 SEQ ID 451-C06 3.0 1.37 108%  86% NO:386 SEQ ID 445-E11 3.7 1.43 69% 79% NO: 387 CLASS VIII SEQ ID 546-G0216.1 1.27 32% 19% NO: 390 SEQ ID 333-C03 12.4 1.37 52% 43% NO: 391 SEQID 549-G05 23.7 1.47 28% 21% NO: 392 SEQ ID 546-B01 8.4 1.20 95% 77% NO:393 SEQ ID 551-D02 13.4 1.37 91% 70% NO: 394 SEQ ID 334-F05 13.5 1.4058% 29% NO: 395 SEQ ID 330-G02 7.4 1.30 37% 31% NO: 396 SEQ ID 316-F087.0 1.30   72% - 38% NO: 397 SEQ ID 332-H09 6.2 1.30 50% 43% NO: 398 SEQID 545-H12 11.3 1.30 74% 60% NO: 399 SEQ ID 548-G05 6.1 1.30 110%  47%NO: 400 SEQ ID 547-C09 4.3 1.23 50% 32% NO: 401 SEQ ID 545-F04 5.2 1.17143%  114%  NO: 402 SEQ ID 552-F06 11.1 1.23 82% 32% NO: 403 SEQ ID531-E11 3.4 1.30 61% 33% NO: 404 CLASS XI SEQ ID 525-A07 7.0 1.17 93%88% NO: 449 SEQ ID 528-F05 4.3 1.10 84% 81% NO: 450 SEQ ID 524-E09 8.21.33 100%  93% NO: 451 SEQ ID  96-H12 35.3 1.37 88% 64% NO: 452 SEQ ID118-A08 11.3 1.30 85% 74% NO: 453 SEQ ID  94-E08 8.9 1.23 102%  74% NO:454 SEQ ID 119-F06 8.0 1.33  4% 27% NO: 455 SEQ ID  95-A11 7.0 1.30109%  108%  NO: 456 SEQ ID  94-H04 7.0 1.37 150%  101%  NO: 457 SEQ ID 94-F07 6.1 1.20 106%  104%  NO: 458 SEQ ID 103-G08 5.7 1.33 140%  95%NO: 459 SEQ ID 118-C07 5.6 1.27 100%  84% NO: 460 SEQ ID 104-C09 5.01.30 64% 50% NO: 461 SEQ ID 117-F08 4.5 1.27 102%  270%  NO: 462 SEQ ID 76-D09 4.4 1.23 79% 87% NO: 463 SEQ ID  93-C08 4.4 1.37 101%  96% NO:464 SEQ ID  92-B05 4.3 1.20 94% 94% NO: 465 SEQ ID 116-H02 4.0 1.23 84%72% NO: 466 SEQ ID  02-B08 3.9 1.30 84% 96% NO: 467 SEQ ID 117-F03 3.81.40 104%  93% NO: 468 SEQ ID 127-A07 3.8 1.20 101%  107%  NO: 469 SEQID  94-B08 3.8 1.20 111%  121%  NO: 470 SEQ ID 115-G02 3.7 1.27 59%  0%NO: 471 SEQ ID 130-E10 3.7 1.80 100%  92% NO: 472 SEQ ID 136-D01 3.71.23 85% 149%  NO: 473 SEQ ID  15-D02 3.6 1.23 97% 118%  NO: 474 SEQ ID 79-B02 3.5 1.30 102%  86% NO: 475 SEQ ID  94-A06 3.5 1.17 84% 96% NO:476 SEQ ID  94-G02 3.5 1.30 108%  76% NO: 477 SEQ ID  75-B12 3.4 1.2395% 108%  NO: 478 SEQ ID 117-F04 3.3 1.37 93% 91% NO: 479 SEQ ID 151-B083.3 1.23 102%  368%  NO: 480 SEQ ID 117-E09 3.3 1.37 109%  102%  NO: 481SEQ ID  93-B10 3.1 1.20  0%  0% NO: 482 SEQ ID  98-F05 3.1 1.23 88% 57%NO: 483 SEQ ID 118-B12 3.1 1.30 98% 112%  NO: 484 SEQ ID  27-D10 3.01.17 111%  131%  NO: 485 SEQ ID 122-D07 3.0 1.63 102%  92% NO: 486 SEQID 149-E06 3.0 1.80 80% 86% NO: 487 SEQ ID 166-H04 3.0 1.27 77% 85% NO:488 SEQ ID  96-D06 3.0 1.37 154%  151%  NO: 489 SEQ ID 103-C04 3.0 1.4073% 86% NO: 490 SEQ ID 527-E08 3.2 1.23 98% 95% NO: 491 SEQ ID 524-H023.2 1.53 26% 25% NO: 492 SEQ ID 523-A04 5.5 1.30 133%  143%  NO: 493 SEQID 524-D07 3.9 1.23 105%  104%  NO: 494 SEQ ID 522-H03 4.5 1.17 107% 94% NO: 495 SEQ ID 527-A10 3.8 1.30 84% 78% NO: 496 Note: Protein ELISAswere measured as fold over background (cMet-Fc vs. TRAIL-Fc) Whole CellELISAs were measured as fold over background (3T3 cells expressing humancMet vs. non-expressing 3T3 cells) HGF competition ELISA measured as a %of binding in the absence of HGF.

TABLE 8 Fluorescence polarization analysis of select peptides from firstgeneration peptide library positive hits SEQ ID NO: Isolate Kd (human)Kd (mouse) CLASS I SEQ ID NO: 001 571-C05 0.20 3.50 CLASS III SEQ ID NO:048 325-H05 3.50 NT SEQ ID NO: 051 336-G04 3.20 NT SEQ ID NO: 052334-G06 2.70 NT SEQ ID NO: 053 330-B07 2.90 NT SEQ ID NO: 055 331-G040.90 1.10 SEQ ID NO: 056 548-F06 2.70 NT SEQ ID NO: 059 323-A11 4.30 NTSEQ ID NO: 061 329-D02 5.20 NT SEQ ID NO: 067 334-C08 1.65 NT SEQ ID NO:068 333-C05 2.80 NT SEQ ID NO: 071 330-G09 1.85 NT SEQ ID NO: 072331-F01 0.98 NT SEQ ID NO: 074 335-D11 3.30 NT SEQ ID NO: 078 331-G062.90 NT CLASS V SEQ ID NO: 369 548-F07 0.88 NB SEQ ID NO: 370 551-H100.22 NB CLASS VIII SEQ ID NO: 390 546-G02 1.50 NT SEQ ID NO: 391 333-C031.80 NT SEQ ID NO: 399 545-H12 1.15 NB CLASS XI SEQ ID NO: 449 525-A076.90 NT SEQ ID NO: 450 528-F05 2.70 NT SEQ ID NO: 451 524-E09 2.00 NTSEQ ID NO: 452  96-H12 >2.00 NT SEQ ID NO: 453 118-A08 >2.00 NT SEQ IDNO: 454  94-E08 0.93 NT SEQ ID NO: 456  95-A11 2.30 NT SEQ ID NO: 458 94-F07 3.75 NT SEQ ID NO: 459 103-G08 >2.00 NT SEQ ID NO: 461104-C09 >2.00 NT SEQ ID NO: 462 117-F08 >2.00 NT SEQ ID NO: 463 76-D09 >2.00 NT SEQ ID NO: 464  93-C08 >2.00 NT SEQ ID NO: 466116-H02 >2.00 NT SEQ ID NO: 467  02-B08 >2.00 NT SEQ ID NO: 469 127-A072.40 NT SEQ ID NO: 472 130-E10 2.60 7.65 SEQ ID NO: 475  79-B02 1.90 NTSEQ ID NO: 479 117-F04 1.70 NT SEQ ID NO: 492 524-H02 0.80 NT Kd valuesare in μM. NB = no binding, NT = not tested

TABLE 9 cMet-binding heteromeric peptide complexes SEQ ID NO: IsolateCLASS PAIR I SEQ ID NO: 472 130-E10 XI SEQ ID NO: 370 551-H10 V PAIR IISEQ ID NO: 369 548-F07 V SEQ ID NO: 370 551-H10 V PAIR III SEQ ID NO:370 551-H10 V SEQ ID NO: 399 545-H12 VIII

TABLE 10 Amino-acid sequence of Mature HSA from GenBank entry AAN17825DAHKSEVAHR FKDLGEENFK ALVLIAFAQY LQQCPFEDHV KLVNEVTEFA KTCVADESAENCDKSLHTLF GDKLCTVATL RETYGEMADC CAKQEPERNE CFLQHKDDNP NLPRLVRPEVDVMCTAFHDN EETFLKKYLY EIARRHPYFY APELLFFAKR YKAAFTECCQ AADKAACLLPKLDELRDEGK ASSAKQRLKC ASLQKFGERA FKAWAVARLS QRFPKAEFAE VSKLVTDLTKVHTECCHGDL LECADDRADL AKYICENQDS ISSKLKECCE KPLLEKSHCI AEVENDEMPADLPSLAADFV ESKDVCKNYA EAKDVFLGMF LYEYARRHPD YSVVLLLRLA KTYKTTLEKCCAAADPHECY AKVFDEFKPL VEEPQNLIKQ NCELFEQLGE YKFQNALLVR YTKKVPQVSTPTLVEVSRNL GKVGSKCCKH PEAKRMPCAE DYLSVVLNQL CVLHEKTPVS DRVTKCCTESLVNRRPCFSA LEVDETYVPK EFNAETFTFH ADICTLSEKE RQIKKQTALV ELVKHKPKATKEQLKAVMDD FAAFVEKCCK ADDKETCFAE EGKKLVAASR AALGL (SEQ ID NO: 647)

TABLE 11 Amino-acid Sequence of SEQ ID NO: 648::HSA::SEQ ID NO: 649GSFFPCWRIDRFGYCHANAP GSGGSGG DAHKSEVAHR FKDLGEENFK ALVLIAFAQY LQQCPFEDHVKLVNEVTEFA KTCVADESAE NCDKSLHTLF GDKLCTVATL RETYGEMADC CAKQEPERNECFLQHKDDNP NLPRLVRPEV DVMCTAFHDN EETFLKKYLY EIARRHPYFY APELLFFAKRYKAAFTECCQ AADKAACLLP KLDELRDEGK ASSAKQRLKC ASLQKFGERA FKAWAVARLSQRFPKAEFAE VSKLVTDLTK VHTECCHGDL LECADDRADL AKYICENQDS ISSKLKECCEKPLLEKSHCI AEVENDEMPA DLPSLAADFV ESKDVCKNYA EAKDVFLGMF LYEYARRHPDYSVVLLLRLA KTYKTTLEKC CAAADPHECY AKVFDEFKPL VEEPQNLIKQ NCELFEQLGEYKFQNALLVR YTKKVPQVST PTLVEVSRNL GKVGSKCCKH PEAKRMPCAE DYLSVVLNQLCVLHEKTPVS DRVTKCCTES LVNRRPCFSA LEVDETYVPK EFNAETFTFH ADICTLSEKERQIKKQTALV ELVKHKPKAT KEQLKAVMDD FAAFVEKCCK ADDKETCFAE EGKKLVAASR AALGLGSGGEGGSG GSWIICWWDNCGSSAP (SEQ ID NO: 650)

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1.-32. (canceled)
 33. A diagnostic imaging agent comprising: a) apolypeptide which binds cMet or which binds a complex comprising cMetand HGF, wherein the polypeptide comprises the amino acid sequence:C-X2-C-X3-G-P-P-X4-F-X5-C-X6-C; wherein X2=H, Y, N, R, S, D, Q, L or E;X3=S, G, N, I, K or Y; X4=T, W, R, S, A or Q; X5=E or Q; X6=W, Y, F, Tor A; and, b) a detectable label for diagnostic imaging.
 34. Thediagnostic imaging agent of claim 33, wherein the polypeptide comprisesthe amino acid sequence:G-X1-C-X2-C-X3-G-P-P-X4-F-X5-C-X6-C-X7-X8-X9-X10-P; wherein X1=S, N, A,K, F, D, I, R, G or T; X2=H, Y, N, R, S, D, Q, L or E; X3=S, G, N, I orY; X4=T, W, R, S, A or Q; X5=E or Q; X6=W, Y, F, T or A; X7=Y or F;X8=G, D, A, E, S, R, H, T or V; X9=T, S, V, M, A, L, Q, E or P; and,X10=E, D, A, N, Y or G.
 35. The diagnostic imaging agent of claim 33,wherein the polypeptide comprises the amino acid sequence:S-C-X2-C-X3-G-P-P-X4-F-X5-C-X6-C-X7-X8-X9-X10; wherein X2=N, H or Y;X3=G or S; X4=T; X5=E; X6=W; X7=Y; X8=A, D, E, G or S; X9=S or T; and,X10=D or E.
 36. The diagnostic imaging agent of claim 33, wherein thepolypeptide comprises the amino acid sequence:E-X12-G-S-C-H-C-S-G-P-α-T-F-E-C-X6-C-X7; wherein X12=A, E, G or S; X6=F,W or Y; and, X7=F or Y.
 37. The diagnostic imaging agent of claim 33,wherein the polypeptide comprises the amino acid sequence:C-Y-C-S-G-P-P-R-F-E-C-W-C orS-E-T-R-P-T-E-A-G-S-C-Y-C-S-G-P-P-R-F-E-C-W-C-Y-E- T-E-P-T-E.


38. The diagnostic imaging agent of claim 33, wherein the detectablelabel is an enzyme, a fluorescent compound, a liposome, an optical dye,a paramagnetic metal ion, an ultrasound contrast agent, or a diagnosticradionuclide.
 39. The diagnostic imaging agent of claim 38, wherein theparamagnetic metal ion or the diagnostic radionuclide is complexed witha chelator.
 40. The diagnostic imaging agent of claim 33, wherein thedetectable label is an ultrasound contrast agent or a diagnosticradionuclide, wherein the diagnostic radionuclide is complexed with achelator.
 41. The diagnostic imaging agent of claim 33, wherein thedetectable label is detectable by optical imaging, acousto-opticalimaging or sonoluminescent imaging.
 42. The diagnostic imaging agent ofclaim 41, wherein the detectable label is a photolabel.
 43. Thediagnostic imaging agent of claim 42, wherein the photolabel is anoptical dye or bioluminescent molecule.
 44. The diagnostic imaging agentof claim 33, further comprising a linker.
 45. A method of detecting cMetor a complex comprising cMet and HGF in a subject, the methodcomprising: (a) administering to the subject the diagnostic imagingagent of claim 33; and, (b) detecting the diagnostic imaging agent inthe subject.
 46. The method of claim 45, further comprising imaging aportion of the subject.
 47. The method of claim 46, further comprisingconstructing an image.
 48. The method of claim 47, wherein the image isconstructed using ultrasound energy, radioimaging or light imaging. 49.The diagnostic imaging agent of claim 37, wherein the detectable labelis an enzyme, a fluorescent compound, a liposome, an optical dye, aparamagnetic metal ion, an ultrasound contrast agent, or a diagnosticradionuclide.
 50. The diagnostic imaging agent of claim 49, wherein theparamagnetic metal ion, or the diagnostic radionuclide is complexed witha chelator.
 51. The diagnostic imaging agent of claim 37, wherein thedetectable label is an ultrasound contrast agent or a diagnosticradionuclide, wherein the diagnostic radionuclide is complexed with achelator.
 52. The diagnostic imaging agent of claim 37, wherein thedetectable label is detectable by optical imaging, acousto-opticalimaging or sonoluminescent imaging.
 53. The diagnostic imaging agent ofclaim 52, wherein the detectable label is a photolabel.
 54. Thediagnostic imaging agent of claim 53, wherein the photolabel is anoptical dye or bioluminescent molecule.
 55. The diagnostic imaging agentof claim 37, further comprising a linker.
 56. A method of detecting cMetor a complex comprising cMet and HGF in a subject, the methodcomprising: (a) administering to the subject the diagnostic imagingagent of claim 37; and, (b) detecting the diagnostic imaging agent inthe subject.
 57. The method of claim 56, further comprising imaging aportion of the subject.
 58. The method of claim 56, further comprisingconstructing an image.
 59. The method of claim 58, wherein the image isconstructed using ultrasound energy, radioimaging or light imaging.